http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Suxiaohui&year=&month=2012.igem.org - User contributions [en]2024-03-28T18:02:13ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Lyon-INSA/contactTeam:Lyon-INSA/contact2012-10-27T03:53:59Z<p>Suxiaohui: </p>
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<h1>Contact us !</h1><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/contactTeam:Lyon-INSA/contact2012-10-27T03:53:24Z<p>Suxiaohui: </p>
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<h1>Contact us !</h1><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/contactTeam:Lyon-INSA/contact2012-10-27T03:53:13Z<p>Suxiaohui: </p>
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/collaborationTeam:Lyon-INSA/collaboration2012-10-27T03:51:55Z<p>Suxiaohui: </p>
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<h1>How did we collaborate?</h1><br />
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Collaboration is a very important issue. We think that sharing our ideas could be very beneficial for both teams engaged in the collaboration. There are more than 240 teams involved in the iGEM 2012 so it was impossible not to find another team sharing some aspects of our project.<br><br />
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<h3>iGEM FRANCE</h3><br />
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<a href="http://sites.google.com/site/igemfrance/"><font>iGEM FRANCE</font></a> is a network gathering the French iGEM community together, linked by a collaborative website. Everything started in February 2012 in Lyon, by the meeting "The iGEM French Touch", organized by the Lyon-INSA iGEM team to help new French iGEMers to improve their scientific, human and financial organization. The ambition of iGEM France is to promote and develop in France SynBio research and development, SynBio teaching, biosafety and bioethics, dialogue between science and society, and develop SynBio programmation by Governmental agencies. <br><br />
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<center><img id="iGEM FRANCE" width="300px" src="https://static.igem.org/mediawiki/2012/9/98/Sans_titre_2.png" alt="iGEM FRANCE"/></center><br />
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<h3>Collaboration with the University of British Columbia (UBC)</h3><br />
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We have established a collaboration with <a href="https://2012.igem.org/Team:British_Columbia">UBC</a> for our Human Practice. Indeed, UBC was working on Intellectual property (IP) this year, an aspect we also chose to explore.<br><br />
One of our advisors is a Professor in Economics. Thus, we have suggested to UBC when they sent their survey on IP, that we could share with them our knowledge and thoughts to improve their survey or to interpret the results of their survey. In exchange they shared the results of their survey with us.<br><br />
We have used their results to show what kind of IP issues iGEM teams could meet.<br />
To see the results of our collaboration you might want to read our <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><font> Human Practice page</font></a>.<br><br />
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<h3>An interesting way to undertake a collaboration with Göttingen team</h3><br />
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Furthermore, we have tried to begin a collaboration with the <a href="https://2012.igem.org/Team:Goettingen">Göttingen team</a>. They are working on a super swimming <i>E. coli</i> strain. We have proposed them different ways for collaboration. For example, we were interested in comparing the impact of swimming on biofilm destabilization between their <i>E. coli</i> strain and our <i>Bacillus subtilis</i> natural swimming strain. We offered to send them our <i>Bacillus subtilis</i> strain to also make them able to perform comparative swimming behavior studies between both strains. Several different reasons made this collaboration unsuccessful.<br><br />
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<h3>Collaboration with TU Munich</h3><br />
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We took part in <a href="https://2012.igem.org/Team:TU_Munich">TU Munich</a>'s survey about their proposition of standardization of BioBricks part descriptions.<br><br />
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<center><img id="TUM12_badge" width="150px" src="https://static.igem.org/mediawiki/2012/c/c6/TUM12_Collaboration_medal1.png" alt="We completed TU Munich's survey on Standardization of BioBrick part descriptions"/></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/HPTeam:Lyon-INSA/HP2012-10-27T03:46:51Z<p>Suxiaohui: </p>
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<p>Our human practice project includes two themes raising many very different questions.<br />
The first one is about various intellectual property issues. The second theme deals with the relation between scientists and public stake holders.</p><br />
<p>We have chosen to divide the theme "intellectual property" into three parts because it represents the main part of our reflexion and required a longer development. <br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and was made by a reflexion on the economic challenges the synthetic biology industry faces. Then we give some answers through a FAQ to the questions you could have after reading our main intellectual property essay. In the third part, we sum up our collaborative work on the intellectual property with the <a href="https://2012.igem.org/Team:British_Columbia">University of British Columbia iGEM team</a> which consists in a survey about IP issues in iGEM. <br />
In the two last parts, we develop our actions towards the public. </p><br><br />
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<h2>Part I: Intellectual property issues</h2><br />
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<h3>Introduction</h3><br />
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Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements : iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
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Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
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The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Commons' system", which will be discussed further on. We will assume that iGEM members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
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We will most notably refer to both Joseph Stiglitz’s (Nobel Prize in Economics in 2001) and Elinor Ostrom’s (Nobel Prize in Economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
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<h3>The patent system: an efficient economic model ?</h3><br />
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<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
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Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted inventions have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
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A patent is granted:<br><br />
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<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
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<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
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The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even genetically modified bacteria may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 1980s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy).<br><br />
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The goal of this brief study though, is not to discuss this matter.<br><br />
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<div class="petitSsTitre">Why patents have been created?</div><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new era for intellectual property began.<br><br />
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Yet, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
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Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patents’ creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e. for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
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As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
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<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
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For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components. When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17,000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
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Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad Genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
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As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licenses of many expensive patents from the incumbent firms.<br><br />
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Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief summary. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ? Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such as shown in inventions and discoveries of more and better ways and means for instance, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
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This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
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Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing. In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
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<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
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In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation. The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to get involved in a collective reflexion on its contribution to the development of a "synthetic biology commons”.<br />
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<h3>The Commons: an alternative model</h3><br />
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Open source softwares' users have been in even greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies, 92% had used open source softwares during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
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<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license. This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
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<br><br />
<br><br />
The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
<br><br />
<br><br />
However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel Prize in Economics in 2009, broadened the economists' mind on this particular topic.<br />
<br><br />
<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th centuries.<br />
<br><br />
<br><br />
Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches : each community managing a commons fixes its own rules by defining a governance framework and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
<br />
<br><br />
<br><br />
Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits :<br />
<br />
<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
<br />
<li>rules in use are well matched to local needs and conditions</li><br />
<br />
<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
<br />
<li>the right of community members to devise their own rules is respected by external authorities</li><br />
<br />
<li>a system for self-monitoring members’ behavior has been established</li> <br />
<br />
<li>a graduated system of sanctions is available</li> <br />
<br />
<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
<br><br />
Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
<br><br />
The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic values of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
<br><br />
<br><br />
<br />
<h3>Bibliography</h3><br />
<br><br />
<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
<br />
<br />
</div><br />
</div><br />
<br />
<h2>Part II: FAQ</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br><br />
Our adventure in Amsterdam made us realize how abstract our reflexion was. We decided then to deepen it in a more concrete way through this FAQ which was inspired by the very questions we were asked in The Netherlands.<br />
<br><br />
<br><br />
<i>Is it entirely impossible to patent an invention based on a public Part ?</i><br />
<br><br />
<br><br />
First of all, we shall remind the patent granting requirements :<br />
<br><br />
<ul><br />
<li>the novelty condition (i.e. the invention must be partially or totally new);</li><br />
<li>the non-obviousness condition (U.S. patent law) or the inventiveness condition (in European patent law);</li><br />
<li>the usefulness condition (U.S. patent law) or the industrial applicability condition (in European patent law).</li><br />
</ul><br />
<br><br />
<br />
<br />
Obviously, patenting public parts is impossible considering the first condition. However novel devices using public parts may be considered for protection through patents, depending on the degree of novelty carried by the device.<br />
<br><br />
<br><br />
<br />
<i>Could private actors like companies belong to and participate to a “synthetic biology commons” ?</i><br><br><br />
<br />
Fact : companies can take part in a commons. Many companies (Small and Medium Enterprises as well as big companies) currently do make profits in the open source software industry. This opportunity is not confined to the software industry : the idea of an “open source biology” has also emerged. This movement, also referred to as “open access biology”, has appeared in bio-industries such as the pharmaceutical sector, in most cases to prevent other companies to patent their research. The SNP Consortium, founded in 1999, gathers 10 pharmaceutical companies which decided to release some of their data concerning the human genome in the public domain. A few other more recent examples, including Pfizer (2006), Novartis (2007) and Syngenta (2002), tend to show that sharing knowledge has become part of the economic strategies in biology (Henkel & Maurer, 2007).<br><br />
<br><br />
Besides, such a sharing knowledge strategy allows companies to make good use of free resources, the validity of which has already been controlled by a (potentially) large community. Moreover, many business models may be designed (and have already been implemented to some extent), which are based on indirect profits, such as selling services for example.<br><br />
<br><br />
More generally, the ability of a commons to create value mainly derives from its capacity to link together different kinds of actors (companies, scholars, hackers, etc.) pursuing different objectives : making profits, publishing, or more original motivations, such as "the fun of programming", "the sense of belonging to a common culture, where participants share a common ideology, often characterized by reciprocity" (Henry and Stiglitz, 2010).<br />
<br><br />
<br><br />
<i>Can a commons coexist with other forms of the organization of economic activities (such as the market or the state involvement) inside the same sector ?</i><br />
<br><br />
<br><br />
Nothing opposes the possibility that different modes of coordination co-exist within the same sector. A given firm may share knowledge in a commons perspective, while simultaneously developing market relations. For instance, some software vendors sell proprietary software (“proprietary bricks”) that complement a main open source program (which is free to use) like an option.<br><br />
<br><br />
Existing economic associations such as the BiOS (“Biological innovation for an Open Society”) Initiative of Cambia, suggest that such a combination would be possible in the field of synthetic biology. The BiOS license allows the licensees to use Cambia enabling technology (a few key plant gene transfer patented technologies) royalty-free, on condition that the improvements made to the technology are also made freely available (i.e. they can be used royalty-free by other licensees). Furthermore, the BiOS founders, who claim that they have adapted the “open source” approach to biology (the source code being here the enabling technology), also argue that their licensing device does not lessen the usual incentives to innovate, including the possibility of patenting some products developed from the application of the enabling technology. <br><br />
<br><br />
<br />
<br />
<i>Why try to find our own rules for the iGEM commons ? The open source seems to work great, so shouldn’t we use theirs ?</i><br />
<br><br />
<br><br />
Ostrom put forward a set of generic principles for a commons to be economically viable. One of them is that every single commons has its own rules that are defined by both its actors and the resources that are dealt with.<br />
Not only are the actors of the open source software movement different from the synthetic biology community but source code is also a resource different from the biological parts. These reasons make it hardly possible that the same rules could be applied to both commons, especially on the legal point of view. Indeed, the open source software movement is based on the spreading of open source licenses, which derive from copyright (“copyleft”). Such a solution is generally considered by legal experts as unadapted to biological parts (Henkel & Maurer, 2007). These experts also suggest that an economic model combining patents with legal devices ensuring free knowledge sharing and use would be the best way to the economic development of synthetic biology and to the spreading of innovations.<br />
<br><br />
<br><br />
<br />
<i>Should we not consider that the bases of a synthetic biology commons have already been defined, by several organisms, including the iGEM Foundation itself ?</i><br />
<br><br />
<br><br />
<br />
The iGEM community has already some attributes of a commons. However, we consider that this commons could be better structured and could play a far bigger role in orienting the development of synthetic biology.<br />
The objectives of our initiative are threefold :<br />
<br><br />
<ul><br />
<li>First, leading every member of the iGEM community to be aware of participating to a commons;</li><br />
<li>Second, provoking a debate within the iGEM community to precise its founding rules. The community should collectively deliberate on the definition of a set of rules regarding the rights to access BioBricks, to use them, to control their application, to impose sanctions if necessary, etc.;</li><br />
<li>Third, leading the iGEM community to discuss its place and the role it may play in the development of an overall synthetic biology commons, involving different kinds of actors : other associations such as the BiOS Initiative, public research units and higher education organizations, companies, and so on. Though building an overall synthetic biology commons is far from being an easy task, it seems to be a growing concern for many actors, including public authorities. For instance, the current Minister of Higher Education and Research in France, Geneviève Fioraso, recently wrote a comprehensive Parliamentary Report on the scientific, technological and economic stakes of synthetic biology, which was published a few months before becoming a Minister (Fioraso Report, 2012). This report notably highlights the crucial role of iGEM in the development of synthetic biology.</li><br />
</ul><br />
<br><br />
<br><br />
<em> Special thanks to our faculty advisor O. Brette for his advice and his help on our intellectual property reflexion.</em><br />
<br><br />
<br><br />
<h3>Bibliography</h3><br />
<br><br />
We used all the previous references (q.v. part I), though we added : <br><br />
<ul><br />
<li>Rai A, Boyle J (2007) <em>Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons.</em> PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058</li><br><br />
<li>Bios website : <a href="http://www.bios.net/daisy/bios/home.html">Bios website</a></li><br><br />
<li> Henkel J, Maurer S (2007) <em>The economics of synthetic biology</em>. Mol Syst Biol 3: 117. </li><br><br />
<li> Fioraso G (2012) <em>Les enjeux de la biologie de synthèse</em>. Report for the "Office parlementaire d'évaluation des choix scientifiques et technologiques".<br />
</ul><br />
<br><br><br />
</div><br />
</div><br />
<br />
<h2>Part III: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey. 281 iGEMers had answered the survey when we treated it.<br />
<br><br />
This collaboration enabled us to broaden our reflexion to iGEM teams' issues.<br />
<br><br />
<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly, we studied former iGEMers' experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
<br><br />
Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
<br><br />
Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may result in a patent experience if the materials used are under a proprietary license. <br />
<br><br />
Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers' IP experiences.<br />
<br/><br />
<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what its nature was. The results are that most of them (46%) had already used patented materials before. However, 15% gave up actually using them, which shows how patents can impede research and innovation : getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
<br><br />
The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10% of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
<br><br />
Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
<br><br />
Our sole conclusion on this matter is that the IPR (Intellectual Property Rights) often do affect iGEM projects, even if in general patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
<br />
<br><br />
It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
<br><br />
Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
<br><br />
To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member and / or to a faculty instructor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
<br><br />
<br><br />
<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the Intellectual Property Rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks.<br />
<br><br />
Besides, this survey deepened our main reflexion (see Part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the Intellectual Property Rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
<br><br />
<br><br />
<em>Thanks to UBC team for sharing their survey results and for their friendly collaboration</em><br />
</div><br />
</div><br />
<br />
<br />
<h2>Part IV: Popularization of Science</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
Stereotypes and various pieces of information about science are spread in such a way that people sometimes find it hard to decipher the truth about everything that is said. The public, but also non-biologist scientists, may sometimes have to look for a reliable source to make their own opinion. <br><br />
We are convinced that we have a role to play and have decided to involve ourselves in several actions.<br />
<br><br />
<br><br />
<h3>First, we organized three conferences with cutting-edge experts:</h3><br />
<ul><br />
<li>“Bacterial swimmers can penetrate biofilms, making them vulnerable to destruction.” by Romain Briandet, expert in Surface Hygiene/Bioadhesion in May 2012.<br />
This meeting enabled the Lyon-INSA iGEM team to discover the amazing properties of Bacillus subtilis swimmers and their potential as a tool for biofilm control.</li><br />
<li>“Synthetic Biology : Biotechnologies revival?” by François Képès, national and international SynBio expert in September 2012. François Képès initiated a debate on the paradox that synthetic biology raises. Paradoxically, synthetic biology aims at improving the industrialization of its products with normalization, but also intends to be more creative by setting free from existing constraints.</li><br />
<li>Olivier Brette in September 2012. The stakes of intellectual property, science and innovation were presented and discussed with the assembly of students and staff from various scientific fields (mechanics, informatics, economic intelligence, ethics…).</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Then, we organized meetings with non-biologist scientists and the public:</h3><br />
<ul><br />
<li>From May to September 2012 : Open debates on the stakes of SynBio with staff and students from Mechanics, Informatics and Telecoms INSA departments but also staff from INSAVALOR (Research and Development, Research Valuation of INSA-affiliated company) through the “Filière Ingénieur-Entreprendre” (Entrepreneurship formation). Participation to the iGEM Entrepreneurship Division was first considered, but the rules were not clear enough to motivate these non biologists to participate to the iGEM competition.</li><br />
<li>September 2012 : The Researchers' Night. Since 7 years, this event has been taking place on a single September night in about 300 cities all over Europe. The main goal of this night is to put researchers in touch with the public. Thus, they can explain what they are doing, how and what can be the applications in the day-to-day life.</li><br />
</ul><br />
<br><br />
<br><br />
<h3>More about the Researchers' Night</h3><br />
This year, the theme was "Imagine the future": what future will emerge from research laboratories? How do scientists imagine the future? Archeology, Physics, Philosophy, Biosciences or Linguistic: researchers shared their lab experiences and thoughts on the future. Therefore, in order to defend a controversial discipline, Synthetic Biology (a.k.a. SynBio), the Lyon-INSA team members participated in the Researchers’ Night, which was held on September 28th, 2012, at the CCO of Villeurbanne.<br />
<br><br />
<br><br />
Indeed, an article published by a French team: “Séralini et al., Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize, Food and Chemical Toxicology, Volume 50, Issue 11, November 2012, Pages 4221–4231” and also in the paper “Le Monde” on September 25th, 2012, on deleterious effect of transgenic maize on rats, had a dramatic effect on the public opinion in Europe. Thus, to illustrate that SynBio can be used to improve the environment, health and life of people, we decided to explain the potential of SynBio and to exchange with the public through a scientific speed-dating and around a table containing diagrams and Petri dishes.<br />
<br><br />
<br><br />
“What is it?” asked a little boy. “What are you doing?” asked a woman. We could introduce them to our friends: bacteria! Then, one of the iGEM team members answered: “Bacteria are little organisms which grow, eat and die like every living being. These bacteria are not always pathogenic and scientists can improve their qualities with the introduction of DNA fragments containing genes.”<br />
<br><br />
<br><br />
So the objective is reached: we aroused the surprise, the questioning and the amazement of people. In this respect, we pointed out the usefulness of SynBio. We decided to focus on medical application of BABS (Bacteria Improved by SynBio) such as insulin, growth hormones, and artemisinin production, and also on the food industry application with food coloring or artificial flavor production that already work.<br />
<br><br />
<br><br />
Of course, we have also presented our project as an innovative solution to kill and disperse biofilm in all pipelines or tanks and to prevent any colonization of bacteria.<br />
<br><br />
<br><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/f/f2/IMG_1082.JPG" class="fancyable"><br />
<center><img src="https://static.igem.org/mediawiki/2012/f/f2/IMG_1082.JPG" width="45%" style="border:5px solid white;vertical-align:top;<br />
"/></center></a><br />
</div><br />
</div><br />
<br />
<h2>Part V: Public opinion</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
We wondered how the consumers would have reacted if our solution had been used in cleaning processes in food and cosmetic industries. To have an idea of their opinion, we have sent a survey to our school members. We have received about 930 answers in less than a week, proving the interest raised by our subject.<br />
<br><br />
<br><br />
<h3>Studied population </h3><br />
<br><br />
<br><br />
We chose to interview a specific population : scientific students from 18 to 24 years old.<br />
<br><br />
<br> <br />
<center><img src="https://static.igem.org/mediawiki/2012/a/ab/Age1.png" width="50%"/></center><br />
<br><br />
<br><br />
Moreover, their studies, knowledge or interests in biology are different. The survey enabled us to precise a tendency : the use of bacteria in food and cosmetic industries is more accepted by the young population, the future consumers. Furthermore, it gives us an idea about the commercial potential of our solution because it is linked to users' acceptance.<br />
<br><br />
<br><br />
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<h3>What is the public opinion about the use of bacteria in cleaning processes?</h3><br />
<br><br />
<br><br />
First, we were interested in the attention the public gives to the composition of every day life products.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/c/c9/Concerned.png" width="70%"/></center><br />
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<br><br />
In general, ¾ of the population is interested and concerned about what kind of substances is used in production processes. So, it seems relevant to ask them what products they are ready to accept.<br />
Food and cosmetics are the products people are most likely to be in contact with. So it is really important to know the opinion of the larger public on this matter :<br />
<br><br />
<br><br />
<u>About cosmetics :</u><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c5/Comparrsoncosm.png" width="90%"/><br />
<br><br />
<div style="opacity:0.4">r\Remark : the bacteria are indicated as eliminated after cleaning and never in direct contact with the products.</div><br />
<br><br />
<br><br />
<u>About food :</u><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f5/Agecomparison.png" width="90%"/><br />
<br><br />
<div style="opacity:0.4">Remark : the bacteria are indicated as eliminated after cleaning and never in direct contact with the products.</div><br />
<br><br />
<br><br />
As we could expect, the biological substances are the ones that people are more willing to accept, both in food and cosmetic industries.<br />
Moreover, there are not real concerns for the use of living bacteria in these two fields.<br />
<br><br />
<br><br />
The questions of the chemicals and the GMO’s is quite different : they are more accepted in cosmetics than in food (in general, people seem to be more careful on what they eat...).<br />
The most remarkable is that the interviewed people have almost the same “reluctance” for the use of chemicals and GMOs. This can be interpreted as the students' sensitization of the negative effects of chemicals.<br />
<br><br />
<br><br />
We find interesting to gather the surveyed ones by age groups : <br />
<br><br />
Some of the surveyed people belong to the 0-17 years old age group, and few to the more than 35 years old. The analysis of their answers lead us to notice that the group above 35 years old has a tendency to refuse GMOs and to accept more easily living bacteria and chemicals in their products. <br />
On the contrary, the youngest ones are more reluctant about chemicals in their foods, and less cautious on what is put in their cosmetics.<br />
<br><br />
<br><br />
Through this survey, we notice the public is ready to accept the use of bacteria in cleaning processes but they seem to be a little bit afraid of GMO's. We have to consider the current French controversy on GMO's which badly influences public opinion. Our meetings with the public showed us that when you take time to really explain how bacterial GMO's are conceived and how they are used, people often change their mind about it. Thanks to these results, we even feel an inducement in our search to limit the use of chemicals and a public approbation of our Biofilm Killer solution.<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/microbialControlTeam:Lyon-INSA/microbialControl2012-10-27T02:13:36Z<p>Suxiaohui: </p>
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<h1>Applications</h1><br />
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<div class="introduction contenuTexte" style="margin:20px;display:inline-block;font-size:15px"><br />
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<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br/><br />
<h2>The versatility of « Biofilm Killer »</h2><br />
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<p>“Biofilm Killer” is designed to meet several industrial needs, because although almost every industry has problems dealing with biofilm development, cleaning biofilms does not mean the same in the different industries.</br><br />
These applications can be briefly categorized in three different needs :</br><br />
<OL><li><i>The need for a clean, microorganism-free and chemical-free surface.</i> The industrial fields requiring this level of protection are basically those dealing with Human and Animal Health issues and in which the presence of contaminating microorganisms (whether good or bad) prevents certification or use of the instruments. This may as well concern the food industry because of health and hygiene related issues, although the use of a probiotic bacterium as a host for Biofilm Killer may be acceptable in certain conditions or countries.<br/><br />
<br/><li><i>The need for a clean deleterious microorganism-free, with a protective bacterial barrier.</i> Two main application domains are concerned. First, in farm animal breeding, protective biofilm barriers are already in use (e.g. poultry), and has application in other animal systems to prevent pathogen infection and excessive mortality. The second application domain is phytoprotection, e.g. biological control of plant pathogens in agriculture. Several microbial agents have been used successfully for years to control plant diseases. In many cases, the biocontrol agent is used to replace the local flora and create a protective barrier against the pathogens to prevent infection. <br/><br />
<br/><li><i>The need for a clean, deleterious microorganism-free but biofilm colinization-protected surface.</i> This may apply to certain areas in which long-term protection would be useful, but the protective biofilm presence is inadequate, e.g. food industry, cosmetics, and other soft-chemical industries. This also apply to refrigeration systems and air-conditioning units.<br />
</OL> <p><br />
<br/><br/>The beauty of Bioflm Killer resides in its versatile design that enables the user to choose the best combination of action for his needs. The 3 modules of Biofilm Killer are independent, and can be used efficiently in any combination with the use of non toxic inducers in very limited quantities.<br/><br />
Basically, given the above classification of needs <strong> the following options are suggested.</strong><br/><br />
</br><br />
<ul><br />
<li><strong>Health and food industry:</strong> Use of only Module 1 (Kill and Scatter) to facilitate complete removal of the biofilm and recover a surface 100% free of bacteria after a regular cleaning procedure step.</li><br />
<li><strong>Poultry and Animal raising, Oil industry :</strong> Use of Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm and induction of Module 3 (STICK) to establish a protective <i>B. subtilis</i> biofilm to create a barrier flora in the intestine of the animal or on the surface of tubing to achieve long-term protection against pathogen recolonization or corrosion.</li><br />
<li><strong>Food industry, Soft chemical industry or the treatment of refrigeration units, air-conditionning modules :</strong> In other applications where the establishement of a bacterial biofilm cannot be envisionned, but long-term protection against microbial recolonization needs to be achieved, the user can use Module 1 (Kill and Scatter) to remove the biofilm in combination with Module 2 (COAT) to generate a protective, peptide-based protective coating of the surface to protect.</li><br/></ul><br />
<br />
To make Biofilm Killer compatible for most industrial applications, we further propose to introduce our genetic constructions in a NON-sporulant <i>Bacillus subtilis</i> strain to prevent the release and survival of this strain in the environment. (<a href="https://2012.igem.org/Team:Lyon-INSA/safety"> See safety page</a>)<br/><br/><br />
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<br />
<h2>Focus on Oil Industry</h2><br />
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<p>The Oil industry faces important and costly difficulties due to biofilm-related issues. These include, but are not limited to, pipeline and metallic structure corrosion, porosity clogging in the rock or the tubing, microbial souring, petroleum spoilage during storage. The main consequences of microbial presence at the global scale are several : <br/> A reduced quality of the final product (microbial alteration), an increase in treatment costs (souring), the reduction of efficient production (souring, microbial alteration, clogging). Biofilm formation is involved in the corrosion of the metallic structures, including the oil-platform, but most importantly the pipelines and tubing. <br/><br/><br />
Biofilm-induced alteration will affect the structure, resulting in two effects : <br/><br />
<ul><br />
<li>The first one is the clogging of the tubing, which much like cholesterol deposits in our arteries, will reduce the available circulating space, reducing the flux of gas or oil that can be transported through the pipe. Even small amounts of biofilm can negatively <br />
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<a href="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" class="fancyable"><br />
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<p>affect flow of hydrocarbons, as can be seen on the figure on the right showing the results of an an experiment performed on gas fluxes in presence or absence of only 8% of biofilm coverage. As a consequence of biofilm formation, we can see that about 50% of the gas flux is lost (<a href="http://www.slb.com/%7E/media/Files/resources/oilfield_review/ors12/sum12/1_microbes.pdf">from Z. Augustinovic <i>et al.</i></a>).</li><br />
<br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<li>The second is the anaerobic corrosion of metal from the structure, which will be instrumental in establishing the biofilm and induce clogging, but will also fragilize the structures.</li><br />
</ul><br />
</p><br />
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<p>In the above example of biofilm-induced pipe corrosion, while a significant part of the corrosion occurs on the outside of the pipe, it is estimated that more than half of the corrosion is due to microbial growth on the inner surface of pipelines. Internal and external metallic corrosion contributes significantly to the risk of oil and gas pipeline deterioration and failure (see below), causes well and reservoir souring and plugging, and results in billions of dollars in annual costs to the oil and gas industry. <br />
</p><br />
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<div class="contenuTexte" style="display:inline-block;width:60%"><br />
<p>Impact of biofilms and microbiologically influenced corrosion in oilfield. Siri platform (center) is located in the North sea and flanked by the smaller Cecilie and Nini satellite platforms. Seafloor lines between the 3 structures and wells carry oil and gas (gas for lift and injection water for pressure support). INSET: in 2007, water injection line ruptured. Subsequent investigation revealed a mixture of iron sulfide and other corrosion by-products plus microbes and polysaccharide slime at the rupture site. These deposits enable sulfate-reducing procaryotes and other troublesome microbes to grow protected from biocides. (Augustinovic <i>et al.</i>, Microbes- Oilfield Enemies or Allies? Oilfield Review, Summer 2012, Schlumberger)</p><br />
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<p>Most importantly, failure on the production line can lead to dramatic oil or gas spills that lead to unprecedented environmental risks and damages. Such failure has recently happened on the Elgin Field, located in the North Sea around 240 kilometers off Aberdeen (Scotland), on a gas field exploited by the French petroleum company Total. The failure of the production line lead to a gas spill estimated to around 1.8 million Euros loss per day for the company. Due to the location of the failure at the sea floor, it took several months for the leak to be stopped. In addition to the cost to the company, a maritime exclusion zone had to be created which perturbated maritime traffic, and around 20 tons of gas were released in the atmosphere daily. The previous year, in the same region an oleoduc exploited by the Royal Dutch Shell had ruptured, also leading to oil spill in the North sea.<br />
</p><br />
<br />
<p>The problems with oil or gas production pipe is two folds : they are expected to be in place for decades and they often are difficult to access. Thus cleaning and locating biofilm is no simple task. Biofilms can be in dead zone which make them impossible to clean with mechanical process.</p><br />
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<h2>« Biofilm Killer »: a practical manual<br />
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<p>Our Biofilm Killer construction in <i>Bacillus subtilis</i> 168 is designed to help address both ease of delivery of the cleaner as well as induce a long-term protection on the inner surface of the tubing. Biofilm Killer can be applied following the procedure which is already accepted in the industrial processes where Clean In Place procedures are performed. In this protocole, the process features 3 tanks usually filled with sodium carbonate, nitric acid and sodium hypochlorite. These three chemicals are used in sequence to remove the biofilm by inducing an alkaline and acidic treatment to destabilize the biofilm and a sterilizing treatment with hypochlorite. Refinements to the CIP procedure include the use of the use of specific enzymes targeting the biofilm, e.g. dispersin (Realco).<br/><br />
<P style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" class="fancyable"><br />
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<p><br>In order to specifically remove biofilms and to lower amounts of toxic chemicals used, we propose to fill one of the CIP bulk with “Biofilm Killer”. “Biofilm Killer” will be targeted to the place to clean by the flow of water delivered in the pipe. It will swim inside the biofilm and produce and deliver <i>in situ</i> inside the biofilm both the biocide and the scattering agent. “Biofilm Killer” will have an action on both the target strain to kill it and on the exopolysaccharide matrix of the biofilm to dissolve it. Our physiological tests show that after 1 hour, there are already significant effects of lysostaphin and dispersin. The impressive effect of the combined action of the two proteins on a staphylococcal biofilm is shown <a href="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg">HERE</a>. To maximise the dispersing effect of the construction, we recommend a 5-hour duration treatment with “Biofilm Killer”. The scattered and killed biofilm will then be eliminated by subsequent acid, caustic and sanitizing treatment as in classical CIP, if no recolonization is required. A significant decrease in the needed amount of these chemical is expected. Alternatively, “Biofilm Killer” will be induced to colonize the surface or produce surfactin in order to form a long-term protection against deleterious recolonization of the surface.<br />
<br/><br />
Preparation and storage of Biofilm Killer is not an issue since the very same organism is already produced and stored for poultry breeding or crop plant phytoprotection. In our case, it will be more convenient to prepare Biofilm Killer as a lyophilisate since it can be stored for months until use.<br />
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<h2>Biofilm Killer and the Oil Patch Economy</h2><br />
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<p><br />
The oil patch is a worldwide industry and moreover a multi-billion dollar activity. It has ramifications worldwide. One can find at least one step of the production or the distribution line in every country. From the exploration to the distribution or the transformation in different products and to the consumer, this system is quite linear and can be schematized as follows : <br/><br />
<p style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" width="80%" /><br />
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This work flow can be divided in two types of activities : <br />
<br/><ul><li>The upstream operations concerns the production <i>sensu largo</i> of the resource itself. It includes all the operations needed for the exploration, the drilling, and the extraction necessary for the crude oil and the natural gas production. </li><br />
<li>The downstream operations deal with the transport and transformation of the product itself. It includes transporting the oil or gas by pipes or tankers, the refining, the retailing and the consuming, until it finally reaches the consumer, which can be the car owner at the gas station or a company using petroleum-derived products.</li><br />
</ul><br />
<br/>The gas and oil patch is the biggest industry in the world. The daily production of oil is around 13 billion liters. It is dominated by 5 or 6, extremely big companies called “Supermajors” which rule the system and have control over it: among them are BP, Total or Shell. Despite their gigantic size, these companies control only around 5% of the oil reserves worldwide. The large majority of the reserve is controlled by local and national companies such as the Saudi Aramco, the iranian National Iranian Oil Company or the koweti Kuwait Petroleum Corporation.<br />
<br/><br/><br />
Oil or gas production involves millions of kilometers of tubing at the production site or for transportation, tanks and tankers for storage and oversea transportation. Moreover, oil extraction often involves the use of large amounts of water, which create a very favorable environments for microbes to grow in. This oil and water mixture travels through the pipe until it is finally separated at the surface. Thus, oil production represents a vast number of tanks and kilometers of pipes in which microbial biofilms are likely to grow and induce corrosion, and which need to be cleaned!<br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png" width="5%" style="float:left;" /><div style="width:800px;float:right;margin-right:10px">This logo means that our solution “Biofilm Killer” can be used in this part of the oil process to remove bacteria and clean pipes or tanks and save the company from much bigger issues, such as a hole in a pipe letting gas or oil flow out in the deep of an ocean, meaning loss of millions dollars.</div><br/><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/BiofilmKTeam:Lyon-INSA/BiofilmK2012-10-27T01:03:48Z<p>Suxiaohui: </p>
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<h1>The Lyon-INSA iGEM 2012 solution: “Biofilm Killer”</h1><br />
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<div class="introduction contenuTexte" style="margin:20px;display:inline-block;margin-top:0"><br />
<div style="float:left;width:600px"><br />
<p>Biological solutions presented previously are very interesting and promising. But synthetic biology could make them even more powerful by providing a technology:<br />
</br><ul><li>able to control, prevent and protect industrial equipment;</li><br />
<li>using non-persistent molecules in the environment;</li><br />
<li>minimizing harm to individual, products and environment.</li></ul></p><br />
<p></br> To meet these challenges, we have chosen to build a bacterial strain based on the environmental friendly <i>Bacillus subtilis</i> strain (already used to feed animals and promote healthy vegetable growth. “Biofilm Killer” was engineered to both destroy and then replace, if needed, the deleterious contamination by a positive biofilm. <br />
</br>Our solution is based on <u><strong>3 genetic modules: </strong></u></p><br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/82/Bacillus.jpg" class="fancyable" style="float:right"><br />
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<p><OL><ul><li><strong>KILL and SCATTER</strong> the biofilm. The effect of the biocide and scattering agents produced by the bacteria can be enhanced by the “torpedo” behavior of the <i>Bacillus</i> swimmers;</li><br />
<li><strong>COAT</strong> the surface with a surfactant reagent in presence of the inducteur 1 (xylose); </li><br />
<li><strong>STICK</strong> to install a positif biofilm in presence of the inducteur 2 (IPTG).</li><br />
</ul><br />
</br> </OL></p><br />
<br />
<h1>Estimating the cost of the current industrial solutions</h1><br />
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<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
In order to compare our biological solution to the currently used ones, we first approached companies whose concerns were similar to ours : they kindly gave us some of their data related to the price of their sterilizing solution.<br><br />
<br />
<p> A first company that accepted to answer our questions estimated that, considering the use of chemical reagents (€50 per use : 3 kg of cleaning products, €15 of disinfectants) and of a sterilizing filter to recover biological waste (€100 to €300 per use depending on its size), the total cost of a 4-hour wash of a 1000L tank was from €165 to €365 ($215 to $475) per sterilization.<br></p><br />
<p> The second one indicated a €480 ($625) cost estimation for a 1500L tank per sterilization.</p><br />
<br>Such estimations made us confident on the competitive potential of our bacteria, considering the low cost of their growth. <br />
<br />
<h1> <a style="text-decoration:none" name="solution"><font color="white">Biofilm Killer implementation : a viable solution</a></font></h1><br />
<br />
<p><br>We extrapolated both our results in 96-well microplates, which showed us the optimal effective concentration of cells for a given surface (2.5x10<sup>8</sup> cells for a growth area of 0.34 cm² covered by <i>S. aureus</i>), and the market prices for genetically modified <i>Bacillus subtilis</i> freeze-dryed cells to an industrial facility model (1000L tank plus 20m of pipes). We used two different prices in our calculations: the average price ($5.55 / kg) and the highest price ($9.58 / kg). <br />
<br>In the end, we estimated that the bacterial cost of the sterilization of our model 1000L facility would be <strong>$0.3 per use</strong> in the first case and <strong>$0.5 per use</strong> in the second one. However, to confine the biological waste, we would also need a double 0.45/ 0.2 µm filter, so that the global price would be increased by at least $100. We also developped a sterilization protocol for this tank (see below) :</p><br />
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<br/><br />
<p>Besides, we generalized our biological prices in relation to a 1 m<sup>2</sup> surface to sterilize : it would then be <strong>$0.06 / m<sup>2</sup></strong> for the average price and <strong>$0.1 / m<sup>2</sup></strong> for the highest one.<br />
<br><br />
"Biofilm Killer" could be conditioned in 25kg bags of freeze-dried cells (usual conditioning in the market) and proposed to sale at an attractive price (estimated $12.25, FOB price).<br />
<br><br />
<br />
<br />
<br />
<h1>Main advantages</h1><br />
</div><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
<p><strong>Lower cost</strong> : No need to have a protein purification step which is very expensive. <br />
<br/><strong>Eco-friendly</strong>: “Biofilm Killer” won’t release toxic chemicals in the environment since the proteins used can be easily destroyed.<br />
<br/><strong>The “biological swiss-knife”</strong>: “Biofilm Killer” should be able to replace mechanical cleaning actions thanks to the swarming properties of our strain which can penetrate deep inside the biofilm and release the active molecules <i>in situ</i> all over the biofilm.</p><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/BiofilmKTeam:Lyon-INSA/BiofilmK2012-10-27T01:03:04Z<p>Suxiaohui: </p>
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<h1>The Lyon-INSA iGEM 2012 solution: “Biofilm Killer”</h1><br />
<br/><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;margin-top:0"><br />
<div style="float:left;width:600px"><br />
<p>Biological solutions presented previously are very interesting and promising. But synthetic biology could make them even more powerful by providing a technology:<br />
</br><ul><li>able to control, prevent and protect industrial equipment;</li><br />
<li>using non-persistent molecules in the environment;</li><br />
<li>minimizing harm to individual, products and environment.</li></ul></p><br />
<p></br> To meet these challenges, we have chosen to build a bacterial strain based on the environmental friendly <i>Bacillus subtilis</i> strain (already used to feed animals and promote healthy vegetable growth. “Biofilm Killer” was engineered to both destroy and then replace, if needed, the deleterious contamination by a positive biofilm. <br />
</br>Our solution is based on <u><strong>3 genetic modules: </strong></u></p><br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/82/Bacillus.jpg" class="fancyable" style="float:right"><br />
<img src="https://static.igem.org/mediawiki/2012/8/82/Bacillus.jpg"; width="300px" style="border:5px solid white;<br />
transform:rotate(7deg);margin-right:10px;margin-top:10px;<br />
-ms-transform:rotate(7deg); /* IE 9 */<br />
-moz-transform:rotate(7deg); /* Firefox */<br />
-webkit-transform:rotate(7deg); /* Safari and Chrome */<br />
-o-transform:rotate(7deg); /* Opera */<br />
"/></a><br />
<div style="clear:both"></div><br />
<br />
<br />
<br />
<br />
<br />
<p><OL><ul><li><strong>KILL and SCATTER</strong> the biofilm. The effect of the biocide and scattering agents produced by the bacteria can be enhanced by the “torpedo” behavior of the <i>Bacillus</i> swimmers;</li><br />
<li><strong>COAT</strong> the surface with a surfactant reagent in presence of the inducteur 1 (xylose); </li><br />
<li><strong>STICK</strong> to install a positif biofilm in presence of the inducteur 2 (IPTG).</li><br />
</ul><br />
</br> </OL></p><br />
<br />
<h1>Estimating the cost of the current industrial solutions</h1><br />
<br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
In order to compare our biological solution to the currently used ones, we first approached companies whose concerns were similar to ours : they kindly gave us some of their data related to the price of their sterilizing solution.<br><br />
<br />
<p> A first company that accepted to answer our questions estimated that, considering the use of chemical reagents (€50 per use : 3 kg of cleaning products, €15 of disinfectants) and of a sterilizing filter to recover biological waste (€100 to €300 per use depending on its size), the total cost of a 4-hour wash of a 1000L tank was from €165 to €365 ($215 to $475) per sterilization.<br></p><br />
<p> The second one indicated a €480 ($625) cost estimation for a 1500L tank per sterilization.</p><br />
<br>Such estimations made us confident on the competitive potential of our bacteria, considering the low cost of their growth. <br />
<br />
<h1> <a style="text-decoration:none" name="solution"><font color="white">Biofilm Killer implementation : a viable solution</a></font></h1><br />
<br />
<p><br>We extrapolated both our results in 96-well microplates, which showed us the optimal effective concentration of cells for a given surface (2.5x10<sup>8</sup> cells for a growth area of 0.34 cm² covered by S. aureus), and the market prices for genetically modified <i>Bacillus subtilis</i> freeze-dryed cells to an industrial facility model (1000L tank plus 20m of pipes). We used two different prices in our calculations: the average price ($5.55 / kg) and the highest price ($9.58 / kg). <br />
<br>In the end, we estimated that the bacterial cost of the sterilization of our model 1000L facility would be <strong>$0.3 per use</strong> in the first case and <strong>$0.5 per use</strong> in the second one. However, to confine the biological waste, we would also need a double 0.45/ 0.2 µm filter, so that the global price would be increased by at least $100. We also developped a sterilization protocol for this tank (see below) :</p><br />
<br />
<br />
<br><center><img src="https://static.igem.org/mediawiki/2012/c/c2/Schema_tank_2.png" width="700px"/></center><br />
<br />
<br/><br />
<p>Besides, we generalized our biological prices in relation to a 1 m<sup>2</sup> surface to sterilize : it would then be <strong>$0.06 / m<sup>2</sup></strong> for the average price and <strong>$0.1 / m<sup>2</sup></strong> for the highest one.<br />
<br><br />
"Biofilm Killer" could be conditioned in 25kg bags of freeze-dried cells (usual conditioning in the market) and proposed to sale at an attractive price (estimated $12.25, FOB price).<br />
<br><br />
<br />
<br />
<br />
<h1>Main advantages</h1><br />
</div><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
<p><strong>Lower cost</strong> : No need to have a protein purification step which is very expensive. <br />
<br/><strong>Eco-friendly</strong>: “Biofilm Killer” won’t release toxic chemicals in the environment since the proteins used can be easily destroyed.<br />
<br/><strong>The “biological swiss-knife”</strong>: “Biofilm Killer” should be able to replace mechanical cleaning actions thanks to the swarming properties of our strain which can penetrate deep inside the biofilm and release the active molecules <i>in situ</i> all over the biofilm.</p><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/BiofilmKTeam:Lyon-INSA/BiofilmK2012-10-27T00:56:09Z<p>Suxiaohui: </p>
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<h1>The Lyon-INSA iGEM 2012 solution: “Biofilm Killer”</h1><br />
<br/><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;margin-top:0"><br />
<div style="float:left;width:600px"><br />
<p>Biological solutions presented above are very interesting and promising. But synthetic biology could make them even more powerful by providing a technology:<br />
</br><ul><li>able to control, prevent and protect industrial equipment<br />
</br><li>using non-persistent molecules in the environment<br />
</br><li>minimizing harm to individual, products and environment</ul></p><br />
<p></br> To meet these challenges, we have chosen to build a bacterial strain based on the environmental friendly <i>Bacillus subtilis</i> strain (already used to feed animals and promote healthy vegetable growth. “Biofilm Killer” was engineered to both destroy and then replace, if needed, the deleterious contamination by a positive biofilm. <br />
</br>Our solution is based on <u><strong>3 genetic modules: </strong></u></p><br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/82/Bacillus.jpg" class="fancyable" style="float:right"><br />
<img src="https://static.igem.org/mediawiki/2012/8/82/Bacillus.jpg"; width="300px" style="border:5px solid white;<br />
transform:rotate(7deg);margin-right:10px;margin-top:10px;<br />
-ms-transform:rotate(7deg); /* IE 9 */<br />
-moz-transform:rotate(7deg); /* Firefox */<br />
-webkit-transform:rotate(7deg); /* Safari and Chrome */<br />
-o-transform:rotate(7deg); /* Opera */<br />
"/></a><br />
<div style="clear:both"></div><br />
<br />
<br />
<br />
<br />
<br />
<p><OL><li><strong>KILL and SCATTER</strong> the biofilm. The effect of the biocide and scattering agents produced by the bacteria can be enhanced by the “torpedo” behavior of the <i>Bacillus</i> swimmers.<br />
</br><li><strong>COAT</strong> the surface with a surfactant reagent in presence of the inducteur 1 (xylose). <br />
</br><li><strong>STICK</strong> to install a positif biofilm in presence of the inducteur 2 (IPTG).<br />
</br> </OL></p><br />
<br />
<h1>Estimating the cost of the current industrial solutions</h1><br />
<br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
In order to compare our biological solution to the currently used ones, we first approached companies whose concerns were similar to ours : they kindly gave us some of their data related to the price of their sterilizing solution.<br><br />
<br />
<p> A first company that accepted to answer our questions estimated that, considering the use of chemical reagents (€50 per use : 3 kg of cleaning products, €15 of disinfectants) and of a sterilizing filter to recover biological waste (€100 to €300 per use depending on its size), the total cost of a 4 hour wash of a 1000L tank was from €165 to €365 ($215 to $475) per sterilization.<br></p><br />
<p> The second one indicated a €480 ($625) cost estimation for a 1500L tank per sterilization.</p><br />
<br>Such estimations made us confident on the competitive potential of our bacteria, considering the low cost of their growth. <br />
<br />
<h1> <a style="text-decoration:none" name="solution"><font color="white">Biofilm Killer implementation : a viable solution</a></font></h1><br />
<br />
<p><br>We extrapolated both our results in 96 microwells plates, which showed us the optimal effective concentration of cells for a given surface (2.5x10<sup>8</sup> cells for a growth area of 0.34 cm² covered by S. aureus), and the market prices for genetically modified Bacillus subtilis freeze-dryed cells to a industrial facility model (1000L tank plus 20m of pipes). We used two different prices in our calculations: the average price ($5.55 / kg) and the highest price ($9.58 / kg). <br />
<br>In the end, we estimated that the bacterial cost of the sterilization of our model 1000L facility would be <strong>$0.3 per use</strong> in the first case and <strong>$0.5 per use</strong> in the second one. However,to confine the biological waste, we would also need a double 0.45/ 0.2 µm filters, so that the global price would be increased by at least $100. We also developped a sterilization protocol for this tank (see below) :</p><br />
<br />
<br />
<br><center><img src="https://static.igem.org/mediawiki/2012/c/c2/Schema_tank_2.png" width="700px"/></center><br />
<br />
<br/><br />
<p>Besides, we generalized our biological prices in relation to a 1 m² surface to sterilize : it would then be <strong>$0.06 / m²</strong> for the average price and <strong>$0.1 / m²</strong> for the highest one.<br />
<br><br />
"Biofilm Killer" could be conditioned in 25 kg bags of freeze-dried cells (usual conditioning in the market) and proposed to sale at an attractive price (estimated $12.25, FOB price).<br />
<br><br />
<br />
<br />
<br />
<h1>Main advantages</h1><br />
</div><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:95%;"><br />
<p><strong>Lower cost</strong> : No need to have a protein purification step which is very expensive. <br />
<br/><strong>Eco-friendly</strong>: “Biofilm Killer” won’t release toxic chemicals in the environment since the proteins used can be easily destroyed.<br />
<br/><strong>The “biological swiss-knife”</strong>: “Biofilm Killer” should be able to replace mechanical cleaning actions thanks to the swarming properties of our strain which can penetrate deep inside the biofilm and release the active molecules <i>in situ</i> all over the biofilm.</p><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/StateArtTeam:Lyon-INSA/StateArt2012-10-27T00:55:22Z<p>Suxiaohui: </p>
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<h1>Biofilm removal: State of the art</h1><br />
<div class="introduction contenuTexte" style="margin:20px;display:inline-block;"><br />
<br />
<div style="text-align:center;"><br />
<a href="https://static.igem.org/mediawiki/2012/d/d4/Encyclo.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Encyclo.jpg" width="200px" style="border:5px solid white;"/></a><br />
<br/><br/><br />
Biofilm removal can be made using different ways, let us introduce you to these techniques.<br />
<br/><br/><br />
<div><center><b><big>Click on the title to show the text.</big></b></center></div><br />
</div><br />
<br/><br />
<h2>Mechanical and Chemical actions</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte" style="display:inline-block;"><br />
<br />
<p>Many strategies and chemical regimens have been defined for controlling biofilms. For open surface in food processing for example, biofilm removal is a multiple step process including <strong>pre-cleaning</strong> by scrapping and rinsing surfaces, <strong>washing</strong> (detergent), <strong>rinsing</strong> (to remove dirt and detergent solutions) and <strong>sanitizing</strong> (to kill attached surviving bacteria and viruses). Closed surfaces such as pipes, tanks or filters require specific equipment such as flooded clean-in-place systems. Flooded systems involve filling all the pipes with successively water, chlorine, biocide, caustic or other chemicals. Other applications use continuous biocide injection procedures to prevent biofilm growth. <br />
</p><br />
<br/><br/><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:60%;"><br />
<p><strong>Mechanical action is often employed to remove biofilms</strong> : If only detergents or sanitizers are used to clean a food line or a pipe in which a biofilm is formed, chemicals contained in the detergent or in the cleaning product can be used to attack and destroy the matrix. But, when the chemical reaches the bacterial colony underlying the matrix, the product may lose its effectiveness in fighting the cells themselves. Scrapping or brushing will unstick the matrix and will expose the underlying bacteria to the action of detergents and sanitizers. Utility pigs allow operators to carry out pipe cleaning.<br />
</p><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/a/ae/Stdprods_utilitypic6.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Stdprods_utilitypic6.jpg" width="300" style="border:5px solid white;<br />
transform:rotate(2deg);vertical-align:top;margin-top:10px;margin-left:20px;<br />
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</a><br />
<br />
<div align="right" style="font-size:13px;margin-right:80px;"><br />
http://www.pipelineengineering.com<br />
</div><br />
<br />
</br><br />
<br />
<br/><br />
<br />
<p>Clean-In-Place (CIP) systems are automatic cleaning system integrated into the machine during its design. A major advantage is that it does not require any system disassembling to operate. The tanks and pipes are cleaned using a parallel fluid circuit. In automated machines, cycles and programs are integrated from the construction. With this system, it is possible to inject the cleaning solution to drain a pipe, but also to include an air- or water-pushed shuttle that scrapes the inner wall of the pipe. In this case, the shuttle is introduced by a parallel circuit or by an aperture in the main pipe. Implementation of an automated CIP system in an existing industrial plant leads to additional costs but is possible (see <a href="http://www.packo.com/en/"> Packo Inox NV </a>).<br />
</p><br />
</br><br />
<br />
<P style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" width="50%" style="border:5px solid white;"/><br />
</a></P><br />
<br />
<br/><br/><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:75%;"><br />
<p>For small industries, more suitable methods have to be considered, such as the injection of water or solutions at high flow, which will cause the detachment of the biofilm due to shear forces. However, some fragments could contaminate the raw material that passes through the pipes like in the dairy farming. It is also possible to increase the temperature. A temperature increase will soften the biofilm but on the other hand, excessively hot temperatures can also lead to other problems such as milk proteins curdling on the milk line surface, thus facilitating the bacteria adhesion.<br />
The products used are terminal types chlorination (chlorine, sodium hypochlorite, monochloramine, chlorine dioxide) for generating a residual biocide. The hydrogen peroxide is also used. Products can be both alkaline and acid coupled with disinfectants.<br />
</p><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/2/2e/Futs-en-plastique-000197913-4.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2e/Futs-en-plastique-000197913-4.jpg" width="20%" style="border:5px solid white;<br />
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</a><br />
<br />
<br/><br />
</div><br />
</div><br />
<br />
<h2>Recent improvement : enzymatic solution</h2><br />
<br />
<div class="wrapper"><br />
<div class="contenuTexte"> <br />
<br />
<div class="contenuTexte" style="width:60%;display:inline-block;"><br />
<br />
<p>As chemical and mechanical actions to clean biofilms are not effective enough, recently (July 2012), the Belgian company <a href="http://www.realco.be/">Realco</a>, working in collaboration with INRA de Lille, specifically with the Laboratoiry Processus aux Interfaces et Hygiène des Matériaux (Processes at Interfaces and Material Hygiene, INRA UR638 PIHM) began to sale their product Biorem in France and in the USA. This alternative method to chemicals for destroying biofilms is based on enzymatic detergents which enables the elimination of biofilms. The Biorem solution contains, among other things, sequestering agents, dispersants, surfactants, stabilizers and enzymes <a href="http://www.realco.be/images/pdf/b2b/BIOREM-A1-BIOREM-10-FR.pdf">(BIOREM A1 + 10)</a>. Recommended for membrane filtration (ultra, micro and nano filtration) or the internal surfaces of CIP systems, this product is expected to eliminate biofilm and ensure optimum output stream, while guaranteeing perfect hygiene of surfaces. The cost of this solution is </p><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/0/0f/Improvement.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0f/Improvement.png" width="30%" style="border:5px solid white;<br />
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"/><br />
</a><br />
<br />
<p>around 500€/m<sup>3</sup> of solution in the case of a CIP use (telephone meeting with the R&D department head of Realco), including the analysis to check the cleaning effectiveness. Realco presents its product as a biofilm destroyer but also as a preventive treatment. This solution is expected to have a lower impact on the environment, with limited release of detergent and total biodegradability, but requires to be used at 50°C to be effective (<a href="http://www.inra.fr/les_recherches/exemples_de_recherche/industries_agroalimentaires_des_enzymes_pour_un_nettoyage_a_fond">INRA website recommendation</a>). <br />
<br/><br />
Another solution to avoid or limit chemicals use, is to prevent the colonization of pathogenic biofilms by spraying a positive bacterial biofilm on surfaces. For example, <a href="http://www.dietaxion.com/bases/produit/pdf1/13/FP_Cobiotex_112_V09_FR_2011-11-04.pdf">the Cobiotex® 112 product</a> , based on a bacterial strain of <i>Bacillus subtilis</i>, is used to limit the development of flora contamination in poultry breeding, and enables animals to evolve in a bio securised area. An other example is the use of <i>Bacillus</i> strains on tomato seeds which promotes a better growth of the crops.<br />
</p><br />
<br />
</div><br />
</div><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/StateArtTeam:Lyon-INSA/StateArt2012-10-27T00:54:10Z<p>Suxiaohui: </p>
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<h1>Biofilm removal: State of the art</h1><br />
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Biofilm removal can be made using different ways, let us introduce you to these techniques.<br />
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<h2>Mechanical and Chemical actions</h2><br />
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<p>Many strategies and chemical regimens have been defined for controlling biofilms. For open surface in food processing for example, biofilm removal is a multiple step process including <strong>pre-cleaning</strong> by scrapping and rinsing surfaces, <strong>washing</strong> (detergent), <strong>rinsing</strong> (to remove dirt and detergent solutions) and <strong>sanitizing</strong> (to kill attached surviving bacteria and viruses). Closed surfaces such as pipes, tanks or filters require specific equipment such as flooded clean-in-place systems. Flooded systems involve filling all the pipes with successively water, chlorine, biocide, caustic or other chemicals. Other applications use continuous biocide injection procedures to prevent biofilm growth. <br />
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<p><strong>Mechanical action is often employed to remove biofilms</strong> : If only detergents or sanitizers are used to clean a food line or a pipe in which a biofilm is formed, chemicals contained in the detergent or in the cleaning product can be used to attack and destroy the matrix. But, when the chemical reaches the bacterial colony underlying the matrix, the product may lose its effectiveness in fighting the cells themselves. Scrapping or brushing will unstick the matrix and will expose the underlying bacteria to the action of detergents and sanitizers. Utility pigs allow operators to carry out pipe cleaning.<br />
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http://www.pipelineengineering.com<br />
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<p>Clean-In-Place (CIP) systems are automatic cleaning system integrated into the machine during its design. A major advantage is that it does not require any system disassembling to operate. The tanks and pipes are cleaned using a parallel fluid circuit. In automated machines, cycles and programs are integrated from the construction. With this system, it is possible to inject the cleaning solution to drain a pipe, but also to include an air- or water-pushed shuttle that scrapes the inner wall of the pipe. In this case, the shuttle is introduced by a parallel circuit or by an aperture in the main pipe. Implementation of an automated CIP system in an existing industrial plant leads to additional costs but is possible (see <a href="http://www.packo.com/en/"> Packo Inox NV </a>).<br />
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<p>For small industries, more suitable methods have to be considered, such as the injection of water or solutions at high flow, which will cause the detachment of the biofilm due to shear forces. However, some fragments could contaminate the raw material that passes through the pipes like in the dairy farming. It is also possible to increase the temperature. A temperature increase will soften the biofilm but on the other hand, excessively hot temperatures can also lead to other problems such as milk proteins curdling on the milk line surface, thus facilitating the bacteria adhesion.<br />
The products used are terminal types chlorination (chlorine, sodium hypochlorite, monochloramine, chlorine dioxide) for generating a residual biocide. The hydrogen peroxide is also used. Products can be both alkaline and acid coupled with disinfectants.<br />
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<h2>Recent improvement : enzymatic solution</h2><br />
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<p>As chemical and mechanical actions to clean biofilms are not effective enough, recently (July 2012), the Belgian company <a href="http://www.realco.be/">Realco</a>, working in collaboration with INRA de Lille, specifically with the Laboratoiry Processus aux Interfaces et Hygiène des Matériaux (Processes at Interfaces and Material Hygiene, INRA UR638 PIHM) began to sale their product Biorem in France and in the USA. This alternative method to chemicals for destroying biofilms is based on enzymatic detergents which enables the elimination of biofilms. The Biorem solution contains, among other things, sequestering agents, dispersants, surfactants, stabilizers and enzymes <a href="http://www.realco.be/images/pdf/b2b/BIOREM-A1-BIOREM-10-FR.pdf">(BIOREM A1 + 10)</a>. Recommended for membrane filtration (ultra, micro and nano filtration) or the internal surfaces of CIP systems, this product is expected to eliminate biofilm and ensure optimum output stream, while guaranteeing perfect hygiene of surfaces. The cost of this solution is </p><br />
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<p>arround 500€/m<sup>3</sup> of solution in the case of a CIP use (telephone meeting with the R&D department head of Realco), including the analysis to check the cleaning effectiveness. Realco presents its product as a biofilm destroyer but also as a preventive treatment. This solution is expected to have a lower impact on the environment, with limited release of detergent and total biodegradability, but requires to be used at 50°C to be effective (<a href="http://www.inra.fr/les_recherches/exemples_de_recherche/industries_agroalimentaires_des_enzymes_pour_un_nettoyage_a_fond">INRA website recommendation</a>). <br />
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Another solution to avoid or limit chemicals use, is to prevent the colonization of pathogenic biofilms by spraying a positive bacterial biofilm on surfaces. For example, <a href="http://www.dietaxion.com/bases/produit/pdf1/13/FP_Cobiotex_112_V09_FR_2011-11-04.pdf">the Cobiotex® 112 product</a> , based on a bacterial strain of <i>Bacillus subtilis</i>, is used to limit the development of flora contamination in poultry breeding, and enables animals to evolve in a bio securised area. An other example is the use of <i>Bacillus</i> strains on tomato seeds which promotes a better growth of the crops.<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/AchievementsTeam:Lyon-INSA/Achievements2012-10-27T00:41:42Z<p>Suxiaohui: </p>
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<h1>Achievements : What did we do ?</h1><br />
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<div> We have <b>registered</b> our team.<br></div><br />
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<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
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<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have set up a <b>team wiki</b>.<br></div><br />
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<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have prepared a <b>poster</b> and a <b>presentation</b> for the Jamboree.<br></div><br />
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<div>We have submitted <a href="https://2012.igem.org/Team:Lyon-INSA/datapage"><b>6 documented parts</b></a> to the registry and showed that they work as expected. Among them, two <b>improvements</b> for existing <i>B. subtilis</i> shuttle vectors : fully characterized plasmids, with the full BioBrick prefix and suffix for the first time in the registry.<br></div><br />
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<div>We have explored <a href="https://2012.igem.org/Team:Lyon-INSA/safety"><b>safety implications</b></a> of the project as a whole.<br></div><br />
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<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have <b>collaborated</b> with <a href="https://2012.igem.org/Team:British_Columbia">British Columbia (UBC) team</a> to exchange knowledge about Intellectual Property. Moreover, we have initiated in february 2012 a french iGEM network and communicate through the <a href="http://sites.google.com/site/igemfrance/">iGEM France website</a>.<br></div><br />
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<div>We have incorporated <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><b>Human Practices</b></a> and <a href="https://2012.igem.org/Team:Lyon-INSA/modelling"><b>Modelling</b></a> into our design.<br></div><br />
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<div>We get our project closer to application by meeting with experts to consider <a href="https://2012.igem.org/Team:Lyon-INSA/Context"><b>industrialization</b></a>.<br></div><br />
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<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have presented our project and Synthetic Biology in many ways : on different <b>websites</b>, in <b>newspapers</b>, in <b>scientific events</b>, in order to inform a public as varied as possible.<br></div><br />
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<div><strong>We have obtained our Gold medal at the Europe Regional Jamboree. </strong><br></div><br />
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<div><strong>Advance to the World Championship Jamboree in Boston. </strong><br></div><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/datapageTeam:Lyon-INSA/datapage2012-10-27T00:40:03Z<p>Suxiaohui: </p>
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<h1>Interactive pattern of our construction : Toggle switch option</h1><br />
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<h1>Our parts : </h1><br />
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<div class="petitSsTitre">Data about our favorite new parts </div><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802001" target="_blank"><font color="#664499"><b>1. Main Page</b></font></a>: <b>Dispersin generator for <i>B. subtilis</i></b> BBa_K802001 : This part associates the <i>Bacillus subtilis</i> constitutive promoter (P<sub>veg</sub>) with the dispersin B gene (<i>dspB</i>). This part allows to efficiently scatter Staphylococci biofilms. <br><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802000" target="_blank"><font color="#664499"><b>2. Main Page</b></font></a>: <b>Lysostaphin generator for <i>B. subtilis</i></b> BBa_K802000 : This part associates the <i>Bacillus subtilis</i> constitutive promoter (P<sub>veg</sub>) with the lysostaphin gene. This part allows an efficient killing of <i>S. aureus</i> cells.<br><br><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802009" target="_blank"><font color="#664499"><b>3. Main Page</b></font></a>: <b>Surfactin generator and biofilm repressor for <i>B. subtilis</i></b> BBa_K802009: This part can be used to induce surfactin production and to repress the biofilm formation in <i> B. subtilis</i> strains (COAT module).<font color="RED"><b>NEW</b></font> <br />
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<div class="petitSsTitre">We've also characterized the following new parts </div><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802002" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>P<sub>lac</sub>-RBS-GFP</b> BBa_K802002 : This part has been designed to determine the behavior of the P<sub>lac</sub> promoter used to drive the STICK module (<span class="unProto" onclick="window.open('http://partsregistry.org/Part:BBa_K802009', 'Part BBa_K802009'); return false;">Part BBa_K802009</span>)<br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802003" target="_blank"><font color="#664499"><b> Main Page</b></font></a>: <b>Shuttle vector for <i>E. coli</i> and <i>B. subtilis</i> </b>BBa_K802003 : This part is a shuttle vector of 6.5kb which is Ampicillin resistant in <i>E. coli</i> and Erythromycin resistant in <i>B. subtilis</i>. It is a <b>low</b> copy number plasmid in <i>B. subtilis</i> and a high copy number plasmid in <i>E. coli</i>. It has a polylinker containing all 4 of the iGEM restriction sites. This vector is characterized in <i>B. subtilis</i> by a transformation yield of 70 transformants/µg and a erythromycin resistance up to 900 µg/mL.<br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802004" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>Shuttle vector for <i>E. coli</i> and <i>B. subtilis</i> </b> BBa_K802004 : This part is a shuttle vector of 6.5kb which is Ampicillin resistant in <i>E. coli</i> and Erythromycin resistant in <i>B. subtilis</i>. It is a <b>high</b> copy number plasmid in both <i>B. subtilis</i> and <i>E. coli</i>. It has a polylinker containing all 4 of the iGEM restriction sites. This vector is characterized in <i>B. subtilis</i> by a transformation yield of 80 transformants/µg and a erythromycin resistance to at least 1.5 mg/mL.<br />
<br><br><br />
<br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802007" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>Biofilm repressor for <i>B. subtilis</i> strains</b> BBa_K802007 : This part can be used to impede biofilm formation in <i>B. subtilis</i>, more particularly if it is arbB-. This part was characterized in conjuction with parts K802006 and K802008.<font color="RED"><b>NEW</b></font> <br />
<br/><br/><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/notebookTeam:Lyon-INSA/notebook2012-10-27T00:15:17Z<p>Suxiaohui: </p>
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<head><br />
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<br />
<div id="xml" style="display:none;"><br />
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<?xml version="1.0" encoding="ISO-8859-1"?><br />
<!-- Edited by XMLSpy --><br />
<project><br />
<month name="July" size="31"><br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 2nd 2012</date><br />
<description>Liquid culture (5 mL LB medium) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description> <br />
</jour><br />
<jour nb="3"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 3rd 2012</date><br />
<description><p>Dilution of 100 µL saturated culture in 5 mL LB medium. </p><br /><br />
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 0.3). </p><br /><br />
Transformation of the NM522 strain (this experiment was repeated 3 times). <br />
<ul><br />
<li>For the positive control the pSB1C3 plasmid was used;</li><br />
<li>For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).</li><br />
</ul><br />
The transformed bacteria were selected on chloramphenicol (Cm) plates.<br />
</description><br />
</jour><br />
<jour nb="4"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 4th 2012</date><br />
<description><br />
Transformation analysis :<br/><br/><br />
<ul><br />
<li>Positive control : lots of colonies;</li><br />
<br />
<li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the two other negative control plates;</li><br />
<br />
<li>Test plate : between 1 and 8 were observed.</li></ul><br />
<br/><br />
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/><br />
<br />
The antibiotic resistance to Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin of the following strains was tested : BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS <i>abrB</i>.<br />
</description><br />
</jour> <br />
<jour nb="5"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 5th 2012</date><br />
<description><br />
Antibiotics resistance tests :<br/><br/><br />
<ul><br />
<li>Bs 168 : no resistance;</li><br />
<li>Bs 168 M cherry : no resistance;</li><br />
<li>Bs 168 GFP : no resistance;</li><br />
<li>Bs <i>abrB</i> : Cm resistant;</li><br />
<li>Bs 168 lysostaphin PWG100 : no resistance;</li><br />
<li><i>S. epidermidis</i> : Tet resistant.</li><br />
</ul><br />
<br/><br />
Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after <i>Eco</i>RI digestion)<br />
<br />
</description><br />
</jour><br />
<jour nb="6"><br />
<titre>For all purposes</titre><br />
<date> Friday, July 6th 2012</date><br />
<description><br />
<br />
Phone conference with Romain Briandet.<br/><br/><br />
<br />
Terminator was retrieved from plate 1 well 13D.<br/><br/><br />
Long meeting.<br />
<br />
</description><br />
</jour><br />
<jour nb="9"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 9th 2012</date><br />
<description><br />
<ul><br />
<li>pBBa_I742123 was put in storage (under the reference pBK1).</li><br />
<li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB medium + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li><br />
<li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li><br />
<li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li><br />
<li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</li><br />
<li>Liquid cultures of the following strains were made : Bs 168 in LB, Bs <i>abrB</i> in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul><br />
<br />
</description><br />
</jour><br />
<jour nb="10"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 10th 2012</date><br />
<description><br />
<ul><br />
<li> The following parts were put in storage :</li><br />
<ul><br />
<li>Lysostaphin in pUC57 Amp resistant (pBK2);</li><br />
<li>Dispersin in pUC57 Amp resistant (pBK3);</li><br />
<li>Surfactin part 2 (RBS-<i>lacI</i>-terminator) in pUC57 Amp resistant (pBK4).</li><br />
</ul><br />
<li> and the following strains (in LB broth supplemented with required antibiotic) :</li><br />
<ul> <br />
<li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li><br />
<li>Bs <i>abrB</i> = BK2 (Cm<sup>R</sup>);</li><br />
<li>Bs 168 = BK3.</li><br />
</ul><br />
<li>Ligation of Dispersin and Lysostaphin biobricks :</li><br />
<ul><br />
<li>digestion of pBK2 with the restriction enzymes <i>Eco</i>RI and <i>SpeI</i>;</li><br />
<li>digestion of pBK3 with the restriction enzymes <i>PstI</i> and <i>XbaI</i>;</li><br />
<li>digestion of pBK4 with the restriction enzymes <i>EcoRI</i> and <i>PstI</i>;</li><br />
<li>3A ligation of the digested parts;</li><br />
<li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2.1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.</li></ul><br />
<li>Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 bands are observed : <strong>PROBLEM. Digestion is made again with E, P and E+P → There are 2 <i>PstI</i> sites !!! WRONG PLASMID</strong></li><br />
<br />
<li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li><br />
<li>Design and order of the primers for the constitutive promoter (part BBa_K143012).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 11th 2012</date><br />
<description><br />
<ul><br />
<li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain.</li><br />
<li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain. <i>(NB : the clones were pink which means that the part contains a RFP gene)</i></li><br />
<li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain.</li><br />
<li>Extraction of pUC57-Lysostaphin/pUC57-Dispersin/pUC57-<i>lacI</i> from transformed NM522 strains.</li><br />
<li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the color of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 12th 2012</date><br />
<description><br />
<ul><br />
<li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li><br />
<li>Reception and storage of the primers (for the amplification of the BBa_K143012 part).</li><br />
<li>The transformed NM522 strain with the part BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit.</li><br />
<li>Extractions with a miniprep kit are made :<br />
<ul><br />
<li>4 clones containing the P<sub>lac</sub> promoter (1,2,3,4);</li><br />
<li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li><br />
<li>3 clones containing theTerminator (1,2,3);</li><br />
<li>4 clones containing the gene for Dispersin;</li><br />
<li>4 clones containing the gene for Lysostaphin;</li><br />
<li>4 clones containing the gene for <i>lac</i>I.</li><br />
</ul><br />
Then a digestion is made on a 0.7% agarose gel.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<titre>Kill</titre><br />
<date> Friday, July 13th 2012</date><br />
<description><br />
<ul><br />
<li>PCR of the constitutive promoter (part BBa_K143012) → the PCR did not work so we ran a new PCR with a more diluted promoter solution.</li><br />
<li>The following strains are put in storage:<br />
<ul><br />
<li>NM522 containing <i>lacI</i>-pUC57;</li><br />
<li>NM522 containing RBS-pUC57;</li><br />
<li>NM522 containing dispersin-pUC57;</li><br />
</ul><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date> Monday, July 16th 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
<ul><br />
<li>PCR of constitutive promoter → it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li><br />
<li>Purification of the PCR product.</li><br />
<li>Gel electrophoresis of lysostaphin.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation of B0015 terminator.</li><br />
<li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date> Tuesday, July 17th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Long morning meeting to discuss results and to write some posters in a frenzied psychopath way in order to remember our tasks.</li><br />
<li>Ligation of promoter-RBS-<i>gfp</i> in plasmid.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168: buffers preparation.</li><br />
<li>Failure of B0015 transformation.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/<i>gfp</i> (of <i>E. coli</i>) using a 3A ligation followed by transformation of the ligation in the NM522 strain;</li><br />
<li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> in 5mL of TSB (conditions described in protocol “Tests on Staphylococcus aureus biofilms in 24 well plate” ).</li><br />
<li>A bacterial suspension of <i>S. epidermidis</i> with OD<sub>600</sub> close to 0.132 is prepared from a Petri dish containing <i>S. epidermidis</i>. A microtiter plate is inoculated with 2 mL of the suspension diluted 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="18"><br />
<date> Wednesday, July 18th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid → extraction failure.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168 (Kit Genomic DNA from tissue).</li><br />
<li>Transformation of pBK5 in <i>B. subtilis</i> <i>abrB</i> failed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation results of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;</li><br />
<li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis : there is still a smear. However, the bands are as expected.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i>.</li><br />
<li>A microtiter plate is inoculated with 2mL of a suspension obtained from diluting the liquid culture 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="19"><br />
<date> Thursday, July 19th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work.</li><br />
<li>A 50µg/mL erythromycin solution is made in order to test the strains’ resistance;</li><br />
<li>Transformation of <i>yfp</i>, <i>gfp</i> and <i>cfp</i> (from iGEM plates) in NM522;</li><br />
<li>B0015 transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth supplemented with Tetracyclin.</li><br />
<li>The fluorescence test of the transformed bacteria containing [dispersin+RBS/<i>gfp</i>+pSB1T3] confirm that the promoter is functional.</li><br />
<li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i> and that the fact of cultivating the strain in broth or on solid medium has no impact on the biofilm's quality.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date> Friday, July 20th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and promoter+pSB1C3 OK → 6 clones are isolated on a LB+Cm plate.</li><br />
<li>Quantification and gel electrophoresis of <i>B. subtilis</i> genomic DNA.</li><br />
<li>Antibiotic testing of <i>B. thuringensis</i> 407 <i>gfp</i> strain and other strains in LB+Ery growth medium.</li><br />
<li>Failure of transforming pBK5 in <i>B. subtilis</i> 168 with the same protocol as previously. New task : find a new protocol.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin.</li><br />
<li>Ligation Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.</li><br />
<li>Ligation Lysostaphin in pSB1C3 and transformation.</li><br />
<li>Isolation of 6 clones of the Terminator transformation.</li><br />
<li>Transformation results of fluorescent genes : ok, except <i>yfp</i>. </li><br />
<li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date> Monday, July 23rd 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Selected clones (NM522+pBK6) are red, meaning that P<sub>veg</sub> promoter and RBS from <i>B. subtilis</i> are recognized by <i>E. coli</i>’s ribosome. 4 clones are streaked in LB+Cm.</li><br />
<li>LB+Ery and TSB+Tet Petri plates are made.</li><br />
<li><i>yfp</i> transformation was successful.</li><br />
<li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li><br />
<li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li><br />
<li>Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results</li><br />
<ul><br />
<li>Promoter+Dispersin in pIG23 : nothing on the plate;</li><br />
<li>Lysostaphin, negative control contains bacteria so the plate is put in junk.</li><br />
</ul><br />
<li>New digestions, ligations,transformations:</li><br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57;</li><br />
<li>Dispersin in pSB1C3;</li><br />
<li>Lysostaphin in pSB1C3;</li><br />
<li>Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).</li><br />
</ul><br />
<li>Streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promoter and the RBS specific to the <i>Bacillus subtilis</i> strain are recognized by the RNA polymerase of <i>E. coli</i>.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR of xylR. Gel electrophoresis : the PCR didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date> Tuesday, July 24th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.</li><br />
<li>Extraction of <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ; test → OK : put in storage.</li><br />
<li>Extraction of the pHT315 GFP plasmid of <i>B. thuringiensis</i> 407 GFP to build a shuttle vector <i>B. subtilis</i> ↔ <i>E. coli</i>. Transformation in NM522 strain and selection on LB+Ampicillin medium : ok.</li><br />
<li><i>S. epidermidis</i> is put in storage in TSB medium under the reference BK15.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results:<br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;</li><br />
<li>Promoter+Dispersin : nothing on the plate;</li><br />
<li>Dispersin in pSB1C3 : a lot of clones;</li><br />
<li>Lysostaphin in pSB1C3 : a lot of clones.</li><br />
</ul><br />
Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (because there are too many clones!)</li><br />
<br />
<li>Extraction of the plasmidic DNA [Promoter in pSB1C3].</li><br />
<li>Extraction of pBK6 from the transformed strain.</li><br />
<li>Extraction of the [Promoter+RBS+<i>gfp</i>] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by <i>Eco</i>RI and <i>Pst</i>I : gel ok. Congrats (pBK12) → 122,6 ng/µL.</li><br />
<li>Extraction of the pBK6 plasmid (P<sub>veg</sub>+RBS+RFP in pSB1C3). The electrophoresis confirms the plasmid’s structure.</li><br />
<li>Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New PCR of xylR with some modifications in the protocol : the PCR didn’t work.<br />
<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date> Wednesday, July 25th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Strains are put in storage :</li><br />
<ul><br />
<li><i>S. epidermidis</i> in TSB+Tet (BK17);</li><br />
<li><i>B. thuringensis</i> 407 GFP in LB+Ery (BK16).</li><br />
</ul><br />
<li>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).</li><br />
<li>Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (GFP or not).</li><br />
<li>The plasmid extraction protocol from <i>B. subtilis</i> was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of the extracted plasmidic DNA of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.</li><br />
<li>Selection of 4 clones transformed by [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.</li><br />
<li>Miniprep to extract [Lysostaphin+Cm] and [Dispersin+Cm] → Digestion.</li><br />
<li>3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).</li><br />
<li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on LB+Tet plates having a biofilm already established and in development of <i>S. epidermidis.</i></li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR on the <i>B. subtilis</i> 168 genome using universal primers of Phil Oger, and positive control with <i>Bulkhoderia cepacia</i> genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of <i>B. subtilis</i> 168. <br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date> Thursday, July 26th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for <i>B. subtilis</i> and <i>E. coli</i>).</li><br />
<li>gDNA extraction with 2 different protocols : the first for <i>Bacillus subtilis</i> 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.</li><br />
<li>PCR of rRNA 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.</li><br />
<li>NM522 + <i>gfp</i>, <i>cfp</i> and <i>yfp</i> in storage.</li><br />
<li>pHT315 GFP put in storage under the reference pBK18.</li><br />
<li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other verification by extracting their plasmidic DNA (but they are not good again).</li><br />
<li>Extraction of the plasmidic DNA of the clones transformed with Lysostaphin+Dispersin in pBK2 and verification by electrophoresis gel. The clones are not right.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A assembly RBS-<i>gfp</i> (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date> Friday, July 27th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
Extraction of transformed clones (NM522/pHT304 and NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites <i>Xba</i>I, <i>Eco</i>RI and <i>Pst</i>I as expected. However, there is a <i>Spe</i>I site which had not been mapped.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.</li><br />
<li>Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.</li><br />
<li>New 3A ligation with Lysostaphin/Terminator/pSB1C3 and transformation in NM522 strain.</li><br />
<li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 and pBK17.</li><br />
<li>Failure of transformation of L1 in NM522.</li><br />
<li>3A assembly of RBS-<i>cfp</i> (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li><br />
<li>3A assembly of <i>sfp</i>-part2(<i>lacI</i>)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>PCR of <i>xylR</i> from gDNA extracted yesterday. Test on gel : successful PCR !!</li><br />
<li>3A ligation of RBS (pBK7) and <i>xylR</i> (produced by PCR) in pSB1C3 ( ! the vector plasmid has the same resistance as the plasmid containing the RBS...).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date> Monday, July 30th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li><strong>It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire…).</strong></li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion test and gel of pBK2 (promoter + lysostaphin) by <i>Eco</i>RI and <i>Spe</i>I, pBK3 (dispersin + terminator) by <i>Xba</i>I and <i>Pst</i>I and pSB1C3 by <i>Eco</i>RI and <i>Pst</i>I.</li><br />
<li>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel → the 3 clones aren’t right.</li><br />
<li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD<sub>600</sub> = 0.5, dilution the culture x100, 1000 and 10 000).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of L1, L2 and IV in NM522.</li><br />
<li>Transformation of <i>sfp</i> and <i>abrB</i> in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Purification of <i>xylR</i>, produced by PCR.<br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date> Tuesday, July 31st 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin).<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i><i>sfp</i></i> parts.</li><br />
<li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of RBS and <i>xylR</i> and transformation in NM522 strain.<br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="August" size="31"><br />
<jour nb="1"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, August 1st 2012</date><br />
<description><br />
<br />
<p>The transformation protocol for <i>Bacillus</i> was tested using the pHT315 GFP plasmid extracted from the <i>B. thuringiensis</i> BT407GFP strain.</p><br />
<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>Lysostaphin+terminator : nothing;</li><br />
<li>Lysostaphin+dispersin : a lot of clones.</li><br />
</ul><br />
Cultures in LB broth of Lysostaphin+Dispersin are launched.</li><br />
<li>Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.</li><br />
<li>pUC57 with Dispersin put in storage : pBK3.</li><br />
<li>Lysostaphin test returns ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The strain NM522/pBK6 was put in storage under the reference BK22.</li><br />
<li>Transformations of the NM522 strain with the ordered constructions (<i>sfp</i> and <i>abrB</i> in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li><br />
<li>Quantification of <i>sfp</i> and <i>abrB</i> constructions provided by Genecust using the Nanodrop.</li><br />
<li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li><br />
</ul><br />
</description> <br />
</jour><br />
<br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Thursday, August 2nd 2012</date><br />
<description><br />
<ul><br />
<li>The transformation of the <i>Bacillus</i> strain failed because of contaminated LB broth, so a new transformation was attempted.</li><br />
<li>pHT304 and pHT315 plasmids supernumerary site <i>Spe</i>I deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dispersin clones digestion : clones not okay.</li><br />
<li>Ligations of :<br />
<ul><br />
<li>Constitutive promoter in Cm iGEM plasmid;</li><br />
<li>Dispersin in Cm iGEM plasmid.</li><br />
</ul><br />
Ligation were verified by electrophoresis.</li><br />
<li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the <i>Bacillus</i> colonies where <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li><br />
<li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the <i>abrB</i> construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li><br />
<li>New ligation of RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <I>sfp</i> and <i>abrB</i> in NM522 strain didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date> Friday, August 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Purification of the NM522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li><br />
<li>The results of <i>Bacillus</i> transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li><br />
<li>A lot of clones for the transformation of the pHT304 and pHT315 plasmids (without <i>Spe</i>I site) on LB+Amp plates → Purification of 12 clones each.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li><br />
<li>Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).</li><br />
<li>BK12 strain verification : spreading on LB + Amp plate.</li><br />
<li>Ligation of Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li><br />
<li>Ligation were verified by electrophoresis.</li><br />
<li>Transformation of Promoter+Dispersin ligation.</li><br />
<li>PCR to check the presence of the promoter in the Promoter in Cm iGEM plasmid clones.</li><br />
<li>Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill <i>Bacillus</i>, 500 µL of Bs 168 pWG100 supernatant, 500 µL of <i>S. epidermidis</i> with OD<sub>600</sub>=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD<sub>600</sub> with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+<i>cfp</i>+pSB1K3).</li><br />
<li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+<i>gfp</i>+pSB1K3).</li><br />
<li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li><br />
<li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New ligation between RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date> Saturday, August 4th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>The negative control is ok;</li><br />
<li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li><br />
</ul><br />
8 clones for each transformation are chosen and spread on LB+Cm and LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformations of <i>abrB</i> and <i>sfp</i> are a success !! :D</li><br />
<li>Liquid cultures of <i>abrB</i> and <i>sfp</i> clones are launched to do plasmid extractions.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Sorting of the RBS + <i>xylR</i> clones to eliminate the clones containing two relegated plasmids.<br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date> Sunday, August 5th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 S.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Plasmid extractions from 3 clones NM522/<i>abrB</i> and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp).<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Liquid cultures of RBS+<i>xylR</i> clones are launched to do plasmid extractions.<br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date> Monday, August 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Plasmid extraction from 6 clones Bs 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li><br />
<li>Miniprep and digestions by <i>Spe</i>I of the plasmid extracted from the 6 clones of NM522 with pHT304 S and NM522 with pHT315 S : Failure ! The <i>Spe</i>I site is still in the plasmids :’( </li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Selection of clones growing on LB+Cm plates and not on LB+Amp plates :<br />
<ul><br />
<li>4 clones Dispersin in pSB1C3;</li><br />
<li>7 clones Promoter pSB1C3;</li><br />
<li>5 clones Promoter+Dispersin in pSB1C3.</li><br />
</ul><br />
Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li><br />
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on LB+Cm and LB+Amp to test their resistance.</li><br />
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of the following ligations, in NM522 strain :</li><br />
<ul><br />
<li>pBK7+pBK13+pSB1K3 (RBS+<i>cfp</i> in Kanamycin resistant backbone);</li><br />
<li>pBK7+pBK14+pSB1K3 (RBS+<i>gfp</i> in Kanamycin resistant backbone).</li><br />
</ul><br />
<li>PCR of RBS-<i>abrB</i> and P<sub>xyl</sub> and purification of these PCR products.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Purification of the <i>xylR</i> gene.</li><br />
<li>Miniprep of RBS-xylR and electrophoresis test → the ligation failed again...</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Tuesday, August 7th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>There is a little plasmid pHT304 S and pHT315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without <i>Spe</i>I site.</li><br />
<li>Transformation in NM522 and spreading on LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li><br />
<li>Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).</li><br />
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li><br />
<li>New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours at 37°C (efficiency of the lysostaphin in stationary phase ?) → the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>3A ligation : [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + [psB1T3].</li><br />
<li>Transformation of the ligation [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + pSB1T3 in NM522 strain and spreading LB+Tet plates.</li><br />
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li><br />
<li>Purification of P<sub>xyl</sub> produced by PCR.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Wednesday, August 8th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 by pHT304 S and pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the <i>Spe</i>I site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...</li><br />
<li>Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.</li><br />
<li>New ligations : <br />
<ul><br />
<li>Promoter+Dispersin in pSB1C3;</li><br />
<li>Lysostaphin+Dispersin in pSB1C3.</li><br />
</ul><br />
Then transformation in NM522.</li><br />
<li>Plasmidic extraction of the BK12 strain : plasmid okay.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Results of the transformations :<br />
<ul><br />
<li>Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li><br />
<li>Transformation by <i>sfp</i>-<i>abrB</i>-pSB1T3 : the negative control isn’t good, the transformation is done again...</li><br />
</ul><br />
<li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Thursday, August 9th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Extraction of NM522/pHT315S. We expected a plasmid without a SpeI site. However, the selected clone contained a plasmid identical to pHT315 (the digestion by <i>Spe</i>I and <i>Eco</i>RI enzymes gives a fragment at 1000 bp). We decided to screen more clones (8 out of 17).</li><br />
<li>The transformation pSB1C3 + RFP and pSB1T3 + RFP is a success ! (red clones)</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
Transformation results : <br />
<ul><br />
<li>Positive control : okay;</li><br />
<li>Negative control : okay;</li><br />
<li>[Lysostaphin + Dispersin] : 8 clones;</li><br />
<li>[Promoter + Dispersin] : more than 30 clones.</li><br />
</ul><br />
Clones are screened on LB + Amp and LB + Cm plates.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The transformation of NM522 strain by <i>sfp</i>-<i>abrB</i> didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the <i>sfp</i>-<i>abrB</i> ligation again...</li><br />
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li><br />
<li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Friday, August 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Deletion of the <i>Spe</i>I site from pHT315 and pHT304 plasmids and filling-in using the <i>Pfu</i> polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li><br />
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.</li><br />
<li>None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.</li><br />
<li>Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.</li><br />
<li>Plasmidic extractions of these clones : clones not okay.</li><br />
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.</li><br />
<li>The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li><br />
<li>The NM522 strain with <i>abrB</i>-pSB1K3 was put in storage under the reference BK25.</li><br />
<li>The NM522 strain with <i>sfp</i>-pSB1K3 was put in storage under the reference BK26.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Saturday, August 11th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Gel electrophoresis showed that the elimination of the <i>Spe</i>I site was not successful.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Sunday, August 12th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Culture of 60 mL of Bs 168 pWG 100 at 28°C for the lysostaphin tests on plates.<br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Monday, August 13th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>We identified the reason why the deletion of the site <i>Spe</i>I from pHT315 and pHT304 plasmids and filling-in was not successful : the <i>Pfu</i> enzyme requires Mg<sup>2+</sup> and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li><br />
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth. These flasks are then frozen at -80°C and freeze-dried overnight.</li><br />
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligation [<i>sfp</i> + <i>abrB</i> + pSB1A3] in the NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Tuesday, August 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The gel electrophoresis of the digested plasmids pHT315 and pHT304 after the final purification shows promising results, so the two plasmids were transformed in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li><br />
<li>Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformation of the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Standard ligation between pBK7 (=RBS in pSB1C3) and xylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain. <br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Wednesday, August 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.</li><br />
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
None of the 4 clones tested had the expected fragments, so we screened 6 others.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by pBK7 and <i>xylR</i> : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + <i>xylR</i> contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and <i>xylR</i> (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Thursday, August 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones having integrated the modified shuttle vector (after filling-in). 4 clones seem to have the right plasmid.</li><br />
<li>The electrophoresis after digestion with <I>Eco</i>RI and <i>Spe</i>I showed that the <i>Spe</i>I site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive promoter (P<sub>veg</sub>) produced by PCR in an iGEM plasmid.</li><br />
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).</li><br />
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.</li><br />
<li>Ligation of P<sub>xyl</sub> produced by PCR in an iGEM plasmid.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Ligation of <i>xylR</i> produced by PCR in an iGEM plasmid (pSB1K3).</li><br />
<li>Results of the transformation of NM522 by [pBK7 + <i>xylR</i>] (for the second ligation) : the plate with bacteria transformed by [pBK7 + <i>xylR</i>] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + <i>xylR</i>]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without <i>xylR</i>.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Friday, August 17th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time, the digestion was successful. The tests showed that there was no <i>SpeI</i> site. However, the pHT315 plasmid also had no XbaI site. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a <i>XbaI</i> site. The digestion of the control plasmid was partial.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Plasmid extractions from the 14 clones transformed by [pBK7 + <i>xylR</i>] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without <i>xylR</i>.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Monday, August 20th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>pBK19 with no SpeI site was put in storage under the reference pBK25.</li><br />
<li>pBK20 with no SpeI site was put in storage under the reference pBK26.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Launch of 8 [Promoter+Dispersin] cultures.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + P<sub>xyl</sub>] : the plate with bacteria transformed by [pSB1T3 + P<sub>xyl</sub>] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + RBS-<i>abrB</i>] : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by [pSB1K3 + <i>xylR</i>] : the plate with bacteria transformed by [pSB1K3 + <i>xylR</i>] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without <i>xylR</i>.</li.<br />
<li>A new PCR of <i>xylR</i> is made, in order to increase the stock.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="21"><br />
<date>Tuesday, August 21st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector <i>E. coli</i> - <i>B. subtilis</i>). Different ligations are made with different proportions of insert and vector. Transformation in NM522 strain.</li><br />
<li>Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.</li><br />
<li>Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li><br />
<li>Results of the second transformation of NM522 by pSB1T3 and RBS-<i>abrB</i> : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Two standard ligations are done :</li><br />
<ul> <br />
<li>pBK7-<i>xylR</i> (<i>xylR</i> cut in X and P sites);</li><br />
<li>pBK24-<i>xylR</i> (<i>xylR</i> cut in E and S sites).</li><br />
</ul><br />
<li>Transformation of pBK7-<i>xylR</i> into NM522 strain.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Wednesday, August 22nd 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 with [Constitutive Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.</li.<br />
<li>New trial of cloning : Digestion, Ligation, transformation to construct :</li><br />
<ul><br />
<li>[Lysostaphin + Dispersin] in pSB1C3;</li><br />
<li>[Lysostaphin + Dispersin] in the shuttle vector (vector for <i>E. coli</i> and <i>B. subtilis,</i>).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 P<sub>xyl</sub> clones were tested, but none had integrated the plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Verification of 6 [<i>xylR</i>-pSB1K3] clones : they only have the vector.</li><br />
<li>Transformation of [pBK24-<i>xylR</i>] into NM522 strain.</li><br />
<li>Results of [pBK7-<i>xylR</i>] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li><br />
<li>Liquid culture of <i>B. subtilis</i> <i>abrB</i> in 5mL of LB broth.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Thursday, August 23rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformations results : All controls are okay.</li><br />
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li><br />
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li><br />
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)</li><br />
<ul><br />
<li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li><br />
<li>The second clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2);</li><br />
<li>The third clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of [P<sub>xyl</sub>+pSB1T3] in NM522.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of [pBK24-<i>xylR</i>] transformation : most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.</li><br />
<li>Extraction of pBK7-<i>xylR</i> from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> <i>abrB</i> in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate').<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Friday, August 24th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A ligation of pBK4 (pUC57 containing the <i>lacI</i> gene) and pBK29 (pSB1A3 containing the <i>sfp</i> and <i>abrB</i> genes) in pBK24 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>After the bad results with <i>xylR</i>, we decided to cut pBK10 plasmid in <i>Sma</i>I site, and ligate with <i>xylR</i>, doing a blunt ligation. We also decided to ligate <i>xylR</i> with <i>xylR</i>, creating a big polymer, which will be like a “pre-ligation” molecule.</li><br />
<li>Extraction of pBK24-<i>xylR</i> from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! Meaning that it’s the first time we manage to ligate <i>xylR</i> successfully.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> <i>abrB</i> strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms.<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date>Saturday, August 25th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of <i>Spe</i>I enzyme, so we could not digest the extracted plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
6 white clones from the ligation pBK24-<i>xylR</i> are purified and put in liquid cultures.<br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date>Sunday, August 26th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Extraction from the 6 white clones (containing <i>xylR</i>-pBK26). Gel electrophoresis is run, but it didn’t show any good clone. <br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date>Monday, August 27th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li><br />
<li>Midiprep : Extraction of P<sub>veg</sub>-dispersin-pSB1C3 of clone 22.</li><br />
<li>BK33 strain was put in storage.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li><br />
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P<sub>xyl</sub>, RBS and <i>sfp</i> gene), <i>abrB</i> gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
No transformation was done, because we ran out of LB broth. <br />
</description><br />
</jour><br />
<br />
<jour nb="28"><br />
<date>Tuesday, August 28th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Meeting at 1:30 pm.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li><br />
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria → Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br /><br />
We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid cultures and then streaked on LB+Cm plates and LB+Amp plates.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Transformation of <i>xylR</i>-pBK10 (blunt end ligation) in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="29"><br />
<date>Wednesday, August 29th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li><br />
<li>Results of the second transformation of Bs 168 strain with pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).</li><br />
<li>We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.</li><br />
<li>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> genes. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.</li><br />
<li>Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using two ligated genes (<i>sfp</i> and <i>abrB</i>) coming from clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classic protocol because we were running out of time and we were behind the schedule.</li><br />
<li>In parallel, we decided to do a standard ligation in order to transfer the <i>lacI</i> gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Transformation of <i>xylR</i>-pBK24 in NM522. The transformation of <i>xylR</i>-pBK24 is done in order to see if <i>xylR</i> was successfully ligated or not. If the bacteria are red, then <i>xylR</i> was not ligated.</li><br />
<li>24 clones containing pBK10-<i>xylR</i> are patched in LB+Amp growth medium, and then incubated.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date>Thursday, August 30th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li><br />
<li>12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.</li><br />
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li><br />
<li>Standard ligation between the shuttle vector pBK26 (pHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.</li><br />
<li>The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.</li><br />
<li>The miniprep confirmed that the ligation containing <i>lacI</i> was successful.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing <i>xylR</i>-pBK10 ligation. The clone is put in liquid culture overnight.</li><br />
<li>Another test is run to confirm that pBK34 plasmid (<i>xylR</i>-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since <i>xylR</i> contains a <i>Nde</i>I restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains <i>xylR</i>. Result : pBK34 doesn’t contain xylem.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date>Friday, August 31st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by <i>Spe</i>I, so the ligation was successful. The four plasmids are further tested for the other 3 iGEM sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digestion was done (<i>Eco</i>RI and <i>Spe</i>I). There was no fragment at 1,000 bp, so the ligation was successful.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li><br />
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li><br />
<li>Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :</li><br />
<ul> <br />
<li>The negative control is ok;</li><br />
<li>The positive control is full of colonies;</li><br />
<li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA. </li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the saturated cultures with transformed bacteria containing the ligation of the <i>sfp</i> and <i>abrB</i> genes. The electrophoresis confirmed that this time the bacteria contained the right plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.</li><br />
<li>The plasmid is digested by <i>Eco</i>RI, <i>Xba</i>I, <i>Spe</i>I and <i>Pst</i>I to find out if the problem comes from a bad site sequence. Result : the <i>Eco</i>RI restriction site is not good, therefore is not recognized by <i>Eco</i>RI enzyme.</li><br />
</ul><br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="September" size="30"><br />
<jour nb="1"><br />
<date>Saturday, September 1st 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the manufacturer protocol and another trial is made with higher concentration.</li><br />
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li><br />
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li><br />
<li>Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into <i>E. coli</i> NM522 strain.</li><br />
<li>6 other clones with the transformed 3A ligation (done on August 29th) are screened.</li><br />
<li>Standard ligation of the <i>abrB</i> gene and the plasmid containing <i>lacI</i> in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="2"><br />
<date>Sunday, September 2nd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated overnight.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized, followed by transformation in <i>E. coli</i> NM522 strain.</li><br />
<li>Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.</li><br />
<li>12 clones for each ligation (done the day before) are screened.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date>Monday, September 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Midiprep of the cloned shuttle vectors (containing the iGEM linker).</li><br />
<li>Miniprep of pBK26 shuttle vector is done in order to increase the stock.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>No clones are obtained on the plate lysostaphin+dispersin when we followed the manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.</li><br />
<li>Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.</li><br />
<li>A transformation is made with our cells of their positive control.</li><br />
<li>Transformations of Bs 168 :<br />
<ul><br />
<li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li><br />
<li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li><br />
</ul><br />
The bacteria are spread on LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).<br /><br />
The transformation of NM522 with ligation Dispersin in pBK26 was successful.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li><br />
<li>6 clones transformed with the mixture of two plasmids are screened.</li><br />
<li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the <i>lacI</i> and <i>abrB</i> genes). The clone is put in storage under the reference pBK39.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date>Tuesday, September 4th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 1.3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.</li><br />
<li>Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.</li><br />
<li>Results of the transformations of Bs 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li><br />
<li>Standard ligations between :<br />
<ul><br />
<li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Pst</i>I;</li><br />
<li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Eco</i>RI.</li><br />
</ul><br />
These two ligation products are transformed in NM522 strain.</li><br />
<li>Acrylamide gels and samples are prepared for SDS-PAGE.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis showed that 5 out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with <i>Eco</i>RV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A PCR is run in order to get RBS-<i>xylR</i>. The PCR failed (not enough DNA-polymerase).<br />
</description><br />
<titre>Biofilm</titre><br />
<description><br />
<ul><br />
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li><br />
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li><br />
<li>Same plate as the one above + crystal violet.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date>Wednesday, September 5th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK22 (<i>sfp</i> gene) and pBK39 (<i>abrB</i>-<i>lacI</i>) and transformation into the <i>E. coli</i> NM522 strain.</li><br />
<li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date>Thursday, September 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD<sub>600</sub> of the liquid cultures is. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Clones obtained on lysostaphin-dispersin plates are put in liquid culture.</li><br />
<li>Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD<sub>600</sub>600 measurements in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The Bs 168 strain is transformed by :<br />
<ul><br />
<li>pBK 25 (= modified shuttle vector pHT304);</li><br />
<li>pBK 26 (= modified shuttle vector pHT315);</li><br />
<li>pBK 28 (= Lysostaphin in the modified shuttle vector pHT304);</li><br />
<li>pBK 38 (= Lysostaphin in the modified shuttle vector pHT315);</li><br />
<li>Dispersin in pBK26.</li><br />
</ul><br />
We spread 200 µL of each transformed cells on LB + Erythromycin ([Ery] = 16 µg/mL) plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones transformed with the ligation pBK22 and pBK39 are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
2 new PCR are done to have <i>xylR</i>, but they failed. <br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Friday, September 7th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.</li><br />
<li>Result of the transformation of Bs 168 : </li><br />
<ul><br />
<li>All the negative control plates are clear : the erythromycin concentration is high enough to do a selection;</li><br />
<li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to extract and analyze their DNA;</li><br />
</ul><br />
<li>Transformation of Bs 168 : new trial by electroporation. As the last transformation, the Bs 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li><br />
<li>A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (<i>sfp</i>, <i>abrB</i> and <i>lacI</i>) would be higher.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A new RBS-<i>xylR</i> PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Saturday, September 8th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Miniprep of the clones Bs 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for <i>E.coli</i> with addition of Lysozyme to the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel → Maybe the DNA isn’t concentrated enough ?<br /><br />
New clones are put in liquid cultures in order to make other minipreps. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of P<sub>lac</sub>-RBS-<i>xylR</i> and then transformation in NM522.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Each of the 24 wells of two microtiter plates are inoculated with 2mL of a suspension obtained from diluting the liquid culture 100 times with TSB enriched with 1% glucose The plate is incubated during 24 hours at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Sunday, September 9th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones Bs 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li><br />
<ul><br />
<li>Bs 168 clone transformed by pBK25 has the right plasmid !! =)</li><br />
<li>Bs 168 clone transformed by pBK28 has the right plasmid !! <strong>BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</strong></li><br />
<li>Bs 168 clones transformed by pBK26 and dispersin in pBK26 : nothing on the gel... </li><br />
</ul><br />
<li>Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + Ery plates, with two different concentrations : [Ery]=10 µg/mL and [Ery]=15 µg/mL.</li><br />
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li><br />
<li>Thanks to the good results, we put in storage the transformed <i>Bacillus</i> under the reference :</li><br />
<ul><br />
<li>BK41 : Bs 168 + pBK25</li><br />
<li>BK42 : Bs 168 + pBK28</li><br />
<li>BK43 : Bs 168 + pBK26 (to confirm)</li><br />
<li>BK44 : Bs 168 + Dispersin in pBK26 (to confirm)</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The supernatant from the two microtiter plates are decanted. The wells of a third microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the dispersin. The supernatants are diluted in order to test different concentrations of the supernatants. The wells of a fourth microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the lysostaphin.<br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Monday, September 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.</li><br />
<li>Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the <i>E. coli</i> NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li><i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.</li><br />
<li>According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD<sub>600</sub> of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD<sub>600</sub>.</li><br />
<li>New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.</li><br />
</ul><br />
</description><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
<ul><br />
<li>Measurement of the NM522 + P<sub>lac</sub> kinetics (with Cm) during 12h in a 96 well plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)</li><br />
<li>In the same 96 well plate NM522 + P<sub>xyl</sub> kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)</li><br />
<li>Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plates prepared the day before are analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can see that the biofilm is not adherent enough and it is destroyed no matter what kind of supernatant is tested. Unfortunately, we cannot say what the effect of the dispersin or lysostaphin is using this kind of test.<br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Tuesday, September 11th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm (plate 2).</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).</li><br />
<li>Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones per transformation are screened.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. <i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.</li><br />
<li>SDS-PAGE is done using pellets, to find a good concentration.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Wednesday, September 12th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation. Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.</li><br />
<li>Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately, the screened clones did not integrate the expected plasmid.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture with the new protocol. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. There is a problem with the results : the control is not good.</li><br />
<li>Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>5 test tubes containing a lamella are inoculated with the culture prepared the day (<i>S. epidermidis</i>) before diluted 1:100 with TSB supplemented with 1% glucose.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Thursday, September 13th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding of 2 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>OD<sub>600</sub> measurements with filtered supernatants.</li><br />
<li>Mobility test of Bs168 on agar 0.25% plate.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.</li><br />
<li>Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Another 12 clones per transformation were screened (from the transformation made on 10th September).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in tanks to test dispersin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce dispersin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. Dispersin has no effect on <i>S. epidermidis</i> culture according to the results.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>One of the tubes and lamella prepared the day before containing <i>S. epidermidis</i> cultures is quantified using crystal violet staining and OD<sub>600</sub> is measured. <i>S. epidermidis</i> cells are quite adherent on glass surfaces.</li><br />
<li>The supernatant of the four other tubes are decanted and tubes are filled with the supernatants produced by the four liquid cultures of <i>B. subtilis</i> prepared the day before in order to test the effect of lysostaphin and dispersin.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Friday, September 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).<br /><br />
Moreover, OD<sub>600</sub> of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.</li><br />
<li>TG1 (<i>E. coli</i>) strain containing pJIM2241 shuttle vector is put in storage (BK47).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600s</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. The strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li>SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The four tubes and lamella prepared the day before are quantified using crystal violet staining and OD<sub>600</sub>is measured. The same result is obtained : <i>S. epidermidis</i> biofilms are not adherent enough and biofilms are destroyed when the growth medium is changed.<br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Saturday, September 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the 2 shuttle vectors pBK35 and pBK36.<br />
</description><br />
<br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in 96 well plate to test impact of lysostaphin and dispersin on a culture of <i>S. epidermidis</i> (test during 12 hours).<br />
</description><br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
LB plates containing a 0.3% agarose gel are inoculated with 5 μL of a 5 hour liquid culture of <i>B. subtilis</i> containing the plasmids used in transformations.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligations between the plasmid pBK42 containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] and the shuttle vectors pBK35 and pBK36 into the NM522 strain .<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Sunday, September 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Screening of 12 clones per <i>Bacillus</i> transformation.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 15 clones transformed with the ligation pBK42+pBK35 and pBK42+pBK36<br />
</description><br />
<br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
The diameter of the disks formed by <i>B. subtilis</i> is measured. The fact of introducing the plasmids did not impact the swarming mobility of our bacteria.<br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Monday, September 17th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Erythromycin at different concentrations was spread on LB plates with in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="18"><br />
<date>Tuesday, September 18th 2012</date><br />
titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened.<br />
</description><br />
<br />
<br />
</jour><br />
<br />
<jour nb="19"><br />
<date>Wednesday, September 19th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.<br />
</description><br />
<br />
<br />
<br />
<br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Thursday, September 20th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector. <br />
</description><br />
<br />
<br />
</jour><br />
<br />
<br />
<br />
<jour nb="21"><br />
<date>Friday, September 21st 2012</date><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
A 7-hour-long kinetics is made with P<sub>lac</sub> and P<sub>xyl</sub> to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used.<br />
P<sub>lac</sub> = IPTG 0.5% and P<sub>xyl</sub> = xylose 0.5%.<br /><br />
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
SDS-PAGE is done in order to detect Dispersin in <i>B. subtilis</i> and <i>E. coli</i>'s supernatants. The gels were not conclusive, and were not good due to an ammonium persulfate problem.<br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Saturday, September 22th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
12 clones transformed with the shuttle vectors are screened.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A new SDS-PAGE is done for dispersin. A migration problem persists, due to ammonium persulfate. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
24 clones transformed with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened.<br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Sunday, September 23th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing 2 mL liquid LB medium are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A last SDS-PAGE is run. This time the problems were successfully solved. But the gels were not conclusive due to a low dispersin concentration.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Monday, September 24th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Both strains grew in all tubes. However, there is a difference concerning OD<sub>600</sub> between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1.5 mg/mL) in order to make sure there is a notable difference between the two strains. </li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<li> In order to characterize the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] two tests are made. To confirm the presence of <i>sfp</i> gene, we made emulsions with the filtered supernatant of the transformed strains and sunflower oil. Concerning <i>abrB</i> gene, a biofilm formation test was made in a 24-well microplate in order to compare the transformed strain to the wild-type strain. <br />
</li><br />
</description><br />
</jour><br />
<jour nb="25"><br />
<date>Tuesday, September 25th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes; <br />
</description><br />
</jour><br />
<br />
</month><br />
<br />
<month name="October" size="31"><br />
<br />
<jour nb="10"><br />
<date>Wednesday, October 10th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation of the part pBK14 containing the <i>gfp</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS).</li><br />
<li>Ligation of the part pBK13 containing the <i>rfp</i> gene with the part pBK7 (containing a <i>B. subtilis</i> RBS).</li><br />
<li>Transformation of the NM522 <i>E. coli</i> strain with the two ligations.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description>Ligation of the PCR product <i>xylR</i> gene with the part pBK9 (P<sub><i>lac</i></sub> gene)and NM522 transformation.<br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Thursday, October 11th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 15 clones per transformation (antibiotic resistance test).<br />
<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Inoculation of 5 mL of LB medium with chloramphenicol with 1 colony of <i>Bacillus subtilis</i> 168 <i>ΔabrB</i> and 5 mL of LB medium with 1 colony of <i>Bacillus subtilis</i> 168. Incubation overnight at 37°C. (This is for the 48h positive <i>Bacillus subtilis</i> biofilm test plate)<br />
</description><br />
</jour><br />
<jour nb="12"><br />
<date>Friday, October 12th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Liquid cultures are inoculated with colonies having the expected antibiotic phenotype.<br />
</br><br />
To test the surfactant properties of the surfactin produced by the <i>B. subtilis</i> BK52 strain, a biofilm test was made in a 24-well plate. Liquid cultures are seeded with BK52 and BK49.<br />
<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>(24h plate)The same inoculation as previously is prepared.</li><br />
<li>(48h plate)<i>Bacillus subtilis</i> 168 <i>ΔabrB</i> or <i>Bacillus subtilis</i> 168 are added in the 12 well plate with MgSO4 1mM and Glucose 0.1 %.</li><br />
</ul><br />
The plate is incubated at 37°C for 48h<br />
<br />
(For both) Inoculation of 5 mL of LB medium with 1 colony of <i>E coli</i> (<i>ompR++</i> GFP, curlis overproduction). Incubation overnight at 37°C. <br />
</description><br />
</jour><br />
<jour nb="13"><br />
<date>Saturday, October 13th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the screened clones. Unfortunately, the gel electrophoresis was disappointing and the extracted plasmids did not contain the ligated genes.<br />
</br><br />
A 24-well plate is incubated with filtered supernatant of the saturated cultures with BK52 and BK49 at room temperature for 24 hours.<br />
<br />
<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
(24h plate)<i>Bacillus subtilis</i> 168 <i>ΔabrB</i> or <i>Bacillus subtilis</i> 168 are added in a 12-well plate with MgSO4 1mM and Glucose 0.1 %.<br />
The plate is incubated at 37°C for 48h.<br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Sunday, October 14th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
The wells are carefully rinsed with M63 medium two times after having previously discarded the supernatant. Then, each well is inoculated with a saturated adherent <i>E. coli</i> strain diluted 100 times in LB medium diluted 2 times.</br><br />
Moreover, another 24-well plate assay was made using the same supernatant, but this time the plate was incubated only 4 hours before <i>E. coli</i> inoculation.<br />
<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
(For both) The supernatant is removed, wells are washed and LB is added.To see if <i>E coli</i> forms a biofilm over the <i>Bacillus subtilis</i> biofilm, the overnight culture of <i>E coli</i> is added into each well.<br />
Plates are incubated for 36h at 30°C.<br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Tuesday, October 16th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately, the observation of the plates under confocal microscope is not possible, so the experiment must be repeated. The only difference is that this time 12-well plates with glass lamellae are used so the observation of the biofilm would be possible.<br />
<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
(For both) Now let's observe using the confocal microscope !<br />
Results : It works ! Our positive <i>Bacillus subtilis</i> biofilm inhibits the stick of other bacteria !<br />
</description><br />
</jour><br />
<jour nb="17"><br />
<date>Wednesday, October 17th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Liquid cultures were inoculated with the strains BK49 and BK52 and then incubated at 37C for 48 hours.<br />
</description><br />
<br />
</jour><br />
<br />
<br />
<jour nb="19"><br />
<date>Saturday, October 19th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
The supernatant from the plate is discarded. Each well is inoculated with an <i>E. coli</i> saturated culture diluted 50 times in LB medium diluted 2 times.<br />
<br />
<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Sunday, October 20th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Ligation of pBK40 and pBK16 followed by transformation into the NM522 strain.<br />
<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="21"><br />
<date>Monday, October 21st 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
The surfactin pre-treated glass lamellae are observed under the confocal microscope. The negative control has a thick biofilm whereas the treated lamella has a few isolated colonies. <br />
<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Tuesday, October 22nd 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 12 clones.<br />
<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Wednesday, October 23rd 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the transformed clones. The gel electrophoresis confirmed the expected insert. (<i>abrB</i> gene in pSB1C3 backbone)<br />
<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Tuesday, October 24th 2012</date><br />
<br />
<titre>Surfactant</titre><br />
<description><br />
The plasmids pBK42 and pBK46 were submitted to the Registry.<br />
<br />
</description><br />
<br />
</jour><br />
<br />
</month><br />
<br />
</project><br />
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<h1>The Notebook</h1><br />
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<h1>Our collections</h1><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/safetyTeam:Lyon-INSA/safety2012-10-27T00:03:46Z<p>Suxiaohui: </p>
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<h1>Safety</h1><br />
<div class="contenuTexte introduction" style="margin:20px"><br />
We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified <i>Bacillus subtilis</i> strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked with an <i>Escherichia coli</i> strain. <i>Staphylococcus epidermidis</i>, <i>S. aureus</i> and adherent <i>E. coli</i> strains were used as biofilm models. <br />
<br><br />
<br><br />
<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br />
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<br />
<h2>Researcher/Public/Environmental Safety</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte" style="display:inline-block;"><br />
<strong>Health and safety training</strong><br> <br><br />
Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider that having a proper training in safety and security at the beginning of our experimental work prepared us to be more organized, responsible for our actions and respectful for those of others. <br />
<br><br />
We have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. No one was allowed to work alone in the laboratory. In addition, the French system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life-saving diploma and are also trained for firefighting. A specific formation for handling chemicals and modified bacteria carrying antibiotic resistance genes was given to each student before starting the bench work.<br />
<br><br><br />
<br />
<strong>Restricted access</strong><br/><br/> <br />
<br />
<div class="contenuTexte" style="width:70%;display:inline-block;"><br />
Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their harmfulness. For these reasons, the access of the laboratory was limited to those involved in the project only.<br />
<br><br />
Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.<br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/8b/Restrictedarea.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8b/Restrictedarea.jpg" width="20%" style="border:5px solid white;<br />
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<strong>Waste management</strong><br/><br/> <br />
Chemical wastes. All reagents were eliminated in the appropriate waste recovery barrels.<br />
<br/><br/><br />
<center><br />
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</a></center><br />
<br><br><br />
<br />
Biological wastes. We used the autoclave of the microbiology teaching platform to decontaminate our biological solid and liquid wastes : no bacteria, modified or not, are released in the environment. <br />
<br />
<br/><br/><br />
<br />
<br />
<div class="contenuTexte" style="width:65%;display:inline-block;"><br />
<strong>Cleaning</strong><br/><br/><br />
All work benches were cleaned every day. The whole lab was cleaned every week.<br />
<br> <br />
CLEAN AREAS TO ENCOURAGE GOOD PRACTICES!<br />
<br/><br/><br />
<strong>Protection</strong><br/><br/><br />
Some of the reagents used are irritant, toxic and can be potential carcinogens (polyacrylamid, Ethidium bromide…). To minimize the impact of their use, they were manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials were clearly identified/labelled to avoid inappropriate mix between two non-compatible solvents.<br />
</div><br />
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<br> <br />
We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates within the double strand DNA helix. To avoid the dissemination of EtBr in the lab, it was stored and used in the same room where the electrophoresis gels were revealed. EtBr was NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. Specific trash barrel for genotoxic contaminated material were used for EtBr-contaminated material.<br />
<br/><br/><br />
To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.<br />
<br><br><br />
<br />
</div> <br />
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<br />
<h2>Specific biological hazards and risks linked to the “Biofilm Killer” project</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br/><br />
<div class="contenuTexte" style="width:65%;display:inline-block;"><br />
<br/><br />
<strong>Bacterial strains</strong><br />
<br/><br/><br />
All <i>Bacillus subtilis</i>, <i>E. coli</i> and <i>S. epidermidis</i> strains we used at INSA-Lyon have a biosafety level of 1, which means that they are not known to cause disease and have minimal environmental hazards. <i>Staphylococcus aureus</i> has a biosafety level of 2 and requires P2 equipment. Manipulations with this organism were conducted in the Bioadhesion/Biofilm lab in Massy (Paris) under Romain Briandet's supervision.<br />
</div><br />
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<div class="contenuTexte" style="width:70%;display:inline-block;margin-top:150px;"><br />
<i>B. subtilis</i> is the chassis chosen for “Biofilm Killer”. <i>B. subtilis</i> is widely used in the field as a biocontrol agent against plant pathogens and food industry processes. It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA). However, if modified by biobricks, <i>B. subtilis</i> has to be physically contained (double filter 0.45/0.2 µm)(<a href="https://2012.igem.org/Team:Lyon-INSA/BiofilmK"> See process in "Our solution"</a>)<br/><br/>. <strong>Enhanced safety can be achieved by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"</strong> <br />
</div><br />
<br/><br />
<i> "The Bacillus subtilis species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1]</i><br />
<i>B. subtilis</i> is known to produce endospores [2] that are resistant to different treatments (heat, UV irradiation…) [3]. Spores of a variety of species are of major concern for the food industry, and even if <i>B. subtilis</i> is non-pathogenic, use of a sporulating strain can be a problem in food industry because spores are resistant to most cleaning procedure and thus can get out of usual control. <br />
Therefore, we plan to introduce a mutation in the <i>B. subtilis</i> strain 168 to disrupt the sporulation process and offer an asporulating version of Biofilm Killer for food application.<br />
<br><br><br />
<br />
<div class="contenuTexte" style="width:70%;display:inline-block;margin-top:50px"><br />
<strong>“Biofilm Killer” power: Lysostaphin, Dispersin, Surfactin</strong><br/><br/><br />
None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: <i>lysostaphin, surfactin and dispersin</i>. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here.<br />
</div><br />
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<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br />
<br>1. European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944. <br />
<br>2. Morphogenesis of Bacillus Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21) <br />
<br>3. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626<br />
<br></div><br />
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</div><br />
<br />
<h2>Biosafety group</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
Our institution (INSA Lyon) has, as every public institute, a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students have followed a 4-hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training though the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies with all French biosafety regulations.<br />
<br/><br/><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/ff/Deathm.jpg" class="fancyable"><img src="https://static.igem.org/mediawiki/2012/f/ff/Deathm.jpg" style="border:5px solid black;border-radius:5px" width="500"/></a></center><br />
<br/><br/><br />
As far as the legal aspect is concerned, there is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is based on the precautionary principle. Even though synthetic biology doesn’t have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.<br />
<br><br />
</div><br />
</div><br />
<br />
<h2>After standard parts, why not a standard chassis ?</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
Beyond traditional <strong>physical containment</strong> (by restricting organisms to a physical space), disabling of the organisms in some way so as to ensure they cannot survive if accidentally or incidentally introduced into the environment. Such disabling leads to <strong>chemical containment</strong> (based on engineering "kill switches" or reliance on nutrients not found in the wild) or <strong>informational containment</strong> (by making genetic information incompatible between natural and synthetic species).<br />
<br><br />
The Lyon-INSA team proposes to provide a “safety kit” to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.<br />
<br><br />
This kit would contain a collection of “disabled chassis” for the most popular chassis (<i>E. coli, B. subtilis</i>…) that could be used to receive the DNA constructions.<br />
<br><br />
As engineering these chassis is not easy, we suggest to create a <strong>new “ Best Safety Device” reward</strong> to motivate iGEM teams.<br />
<br><br><br />
<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br/><br />
1 Contreras A., Molin S, Ramos JL. 1991. Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics. Applied and Environmental Microbiology.<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/modellingTeam:Lyon-INSA/modelling2012-10-26T23:23:44Z<p>Suxiaohui: </p>
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<h1>Modelling</h1><br />
<div class="contenuTexte"><br />
<div style="float:left;width:680px;margin-left:20px;"><br />
<br/><br />
<br/><br />
An interesting question when you are more of a biologist than a mathematician (too many complicated equations!!!). And most of our team members are biologists/biochemists… Thus, we tried to explain modelling and our model in a comprehensive way for everyone.<br/><br/><br />
<br/><br/><br />
<span style="margin-top:150px;margin-left:100px; font-size:20px;">This is our Biological Modelling for Dummies ! </span><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2012/8/82/Dummies.jpg" width="250" style="float:right;margin-right:20px;"/><br/><br />
<div style="clear:both"></div><br />
<br />
<br />
<br />
<br/><br/><br />
<br />
<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br />
<br />
<br />
<h2>What is modelling ?</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Definitions</h3><br />
<br><br />
A <b>model</b> is a symbolic representation of an object’s or a phenomenon’s aspects in the real world.<br/><br />
<br/><br />
<center>“<i>All models are false. Some are useful.</i>” Georges Box</center> <br/><br />
<br/><br />
<b>Modelling</b> is the process that enables the development of a model. It takes into account [1]:<br/><br />
<ul><br />
<li>The phenomenon to represent </li><br />
<br />
<li>A specific formal system (equation, diagram..)</li><br />
<br />
<li>Objectives (what we want to do with the model)</li><br />
<br />
<li>Data (for variables) and knowledge (relation between variables) available or accessible by experimentation or observation</li><br />
</ul><br />
<br><br><br />
<br />
The <b>tasks</b> to obtain and use the model depend on the biological situation and the formal system chosen. Nevertheless, it must:<br/><br />
<ul><br />
<li>Have a formalization work, which is the model writing </li><br />
<br />
<li>Manipulate the model in the formal system to describe its properties (theoretical main behavior regardless of the values of the parameters)</li><br />
<br />
<li>Establish relationships with other representations (computer program, graph function)</li><br />
<br />
<li>Interpret and compare different representations obtained in the formal world with the biological reality (often that reality is seen through experimental data)</li><br />
<br />
</ul><br />
<br/><br />
<div style="font-size:12px"><br />
<b>References: </b><br><br />
<ul><br />
<li> [1] Alain Pavé, <i>Modélisation en biologie et en écologie</i>, Aléas, 1994 <br />
</ul><br />
<br><br />
</div><br />
</div> <br />
</div><br />
<br />
<h2>Biological System description</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<h3>Situation</h3><br />
<br><br />
After the destruction of the biofilm by “Biofilm Killer” bacteria, we want to have the choice to either create a surfactant or establish a positive biofilm, both to prevent the recolonization of the surface by deleterious organisms. The toggle switch is done by environmental conditions : two inducers can be added to select one behavior or another.<br><br />
<br />
<div class="petitSsTitre">Biological system to model</div><br />
For this, we have created the following construction, with a double regulation:<br><br />
<br> <br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d6/2%29_biological_system.png" width = 450px/></center><br><br />
<center><small>Figure 1: The construction of the biological model, with the following elements: 2 promoters (P<sub><i>xyl</i></sub> and P<sub><i>lac</i></sub>), 2 repressors (LacI and XylR proteins) <br>and 2 inducers (IPTG and Xylose), and also <i>sfp</i> and <i>abrB</i> genes for Sfp and AbrB proteins.</i></small></center><br><br />
<br><br />
<br />
This system is a gene-regulatory network, where two different states are possible:<br><br />
<ul><br />
<li><b>Formation of a naturally toxic bio-surfactant through <i>sfp</i> gene</b>, which has antimicrobial properties that prevents the recolonization of the surface. The surfactant used is surfactin, whose production is activated by the <i>sfp</i> gene. This is the <b>COAT</b> option.</li><br />
<br />
<li><b>Establishment of a positive biofilm</b> by the inhibition of the main biofilm repressor gene <i>abrB</i>. This is the <b>STICK</b> option.</li><br />
</ul><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/a/a5/2%29_constructs.png" width=450px/></center><br><br />
<center><small>Figure 2: The two possible states, surfactant formation for the top operon or biofilm formation for the bottom construction.</small></center><br><br />
<br />
<br />
<br />
LacI and XylR proteins are called repressors. They bind to their respective promoters (P<sub><i>lac</i></sub> and P<sub><i>xyl</i></sub>), thus preventing RNA polymerase binding. So proteins under the inactivated promoter are not produced. <br> There are also two inducers in the system: <b>IPTG</b> (isopropyl β-D-1-thiogalactopyranoside) and <b>Xylose</b> (monosaccharide of the aldopentose type). In the absence of these inducers, both constructions are inhibited. If only one of them is present, the corresponding inhibition disappears and the associated construction is expressed.<br><br />
<br><br />
<div style="display:inline-block;margin-right:20px;text-align:center" ><br />
<iframe frameborder="0" width="430" height="280" src="http://www.dailymotion.com/embed/video/xttbqi"></iframe><br /><a href="http://www.dailymotion.com/video/xttbqi_xylose-induction_tech" target="_blank">Xylose Induction</a><br />
</div><br />
<br />
<div style="display:inline-block;text-align:center;float:right;"><br />
<iframe style="display:inline-block" frameborder="0" width="430" height="280" src="http://www.dailymotion.com/embed/video/xttbr9"></iframe><br /><a href="http://www.dailymotion.com/video/xttbr9_iptg-induction_tech" target="_blank">IPTG Induction</a><br />
</div><br />
<br />
<br><br><br />
For example, in the presence of Xylose, XylR proteins will form an enzymatic complex with their Xylose sugar. Thus, the inhibition of P<sub><i>xyl</i></sub> caused by XylR binding will disappear, resulting in an enhanced production of Sfp, AbrB and LacI proteins. Sfp production induces surfactin production, and AbrB production involves the repression of the formation of the biofilm. Eventually, LacI production will inhibit XylR production, <b>so there will be stabilisation of P<sub><i>xyl</i></sub> activation.</b><br><br />
In opposition, in the presence of IPTG, LacI proteins will bind to their ligand, and P<sub><i>lac</i></sub> promoter will be free. So XylR proteins will be overproduced, limiting Sfp and AbrB productions. Thus, there will be no surfactin in the environment, biofilm formation can begin.<br><br />
<br><br />
<br />
<div class="petitSsTitre">Aim of the model:</div><br />
<br />
<br><br />
With this model, we pursue two main objectives : <br><br />
<ul><br />
<li>1) Verify the design of the biological system, to be sure that the toggle switch is functional; </li><br />
<li>2) Predict the behaviour of this biological system depending on the presence of inducers to give usage guidelines for its industrialization.</li><br />
</ul><br><br />
<br />
<b>However...</b><br><br><br />
We are working in a <i>Bacillus subtilis</i> strain and some parameters such as XylR values on binding/unbinding kinetics to both inducer and promoter or production rate from P<sub><i>xyl</i></sub> promoter cannot be found in the literature and most of the existing values come from an <i>E. coli</i> strain rather than <i>B. subtilis</i>. <br />
Furthermore, we are finishing the biological system construction and its characterization is on the way. Parameters will be measured very soon.<br><br />
<br><br />
Because of this lack of information, we will create a theoretical model in order to characterize the global behavior of the system.<br><br />
<br><br />
Moreover, as we are mainly biologists in the team, we thought it could be interesting to explain how we can easily obtain a mathematical model from a biological system. <br> <br />
<br />
<br />
</div><br />
</div><br />
<br />
<h2>Biological modelling for dummies !</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<br><br />
<br />
<h3>Basic knowledge</h3><br />
<br><br />
We want to transform the biological system into mathematical equations in order to be able to determine the quantity of inducers (input) needed to obtain a particular behavior (output).<br><br />
<br />
<br><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/27/3%29_Black_Box.png" border="1"width=550px/></center><br />
<center>Figure 3: The black box model: <br>there will be the STICK or COAT option depending on the inducers concentration</center><br><br />
<br><br />
<br />
<div class="petitSsTitre">Ordinary differential equation (ODE)</div><br />
<ul><br />
<li>mathematical equation;</li><br />
<li>format: <img style="margin-left:20px;" src="https://static.igem.org/mediawiki/2012/4/42/3%29_EDO.png" width=10%/></li><br />
<li>explanation: used in biology and physics to represent the growth or evolution of a quantity dx (i.e. population or concentration) proportional to the population size/effective concentration x during a period of time t;</li><br />
<li>x is called a variable.</li><br />
</ul><br />
<br><br><br />
<h3>Elements of the model</h3><br />
<br><br />
<div class="petitSsTitre">First list of variables</div><br />
We want to have the concentration of LacI and XylR as outputs depending of the inducers concentrations input. We know that the repressors can bind either to their promoter (P<sub><i>lac</i></sub> and P<sub><i>xyl</i></sub> respectively) or to their inducer (IPTG and xylose). Thus, first of all, we have the following variables in the system:<br><br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/5/50/3%29_element_1.png" width=400px/></center><br />
<center>Figure 4: The model variables at first glimpse</center><br><br />
<br><br />
<div class="petitSsTitre">Binding and unbinding kinetics</div><br />
We decided to analyze the relation between repressors, promoters and inducers depending on the law of mass action. It is a branch of chemical kinetics, which states that the speed of a chemical reaction is proportional to the quantity of the reacting substances. These substances will bind with an association kinetic k and unbind with a dissociation kinetic k<sub>m</sub>. </br><br><br />
<br />
<div style="display:inline-block;text-align:center;margin-top:60px"><br />
<iframe frameborder="0" width="430" height="250" src="http://www.dailymotion.com/embed/video/xttbop"></iframe><br />
<br /><br />
<a href="http://www.dailymotion.com/video/xttbop_binding-and-unbinding-kinetic_tech" target="_blank">Binding and unbinding kinetic</a> <br><br />
</div><br />
<br />
<div style="display:inline-block;text-align:center;float:right"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/3%29_binding_and_unbinding.png" width=400px/><br />
<br/><br />
Figure 5: Binding and unbinding kinetics in the model<br />
</div><br />
<br />
<br><br />
<div style="clear:both;"></div><br />
<br>It is working either for LacI binding to its P<sub><i>lac</i></sub> promoter than for XylR to P<sub><i>xyl</i></sub> and also the inducers and LacI and XylR. This binding creates a new complex.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/3/32/3%29_element_2.png" width=450px/></center><br />
<center>Figure 6: The model variables</center><br><br />
<br />
<br />
<h3>Equations of the model</h3><br />
<br />
<br>Now, we can find the equations, based on the behavior of each element. There will be 3 types of equations, each of them related to the nature of the variable, i.e. a promoter, an inducer or a repressor.<br><br />
<br />
<div class="petitSsTitre">Promoters</div><br />
As described above, there are two promoters, P<sub><i>lac</i></sub> and P<sub><i>xyl</i></sub>, and each of them can be free (with no repressor bound on it) or occupied (with the corresponding associated repressor).<br />
<br />
<br>Thanks to the law of mass action and binding and unbinding kinetics, we obtain the equations like this:<br><br><br />
<br />
<div style="display:inline-block;text-align:center;"><br />
<iframe frameborder="0" width="430" height="250" src="http://www.dailymotion.com/embed/video/xttbtn"></iframe><br /><a href="http://www.dailymotion.com/video/xttbtn_plac-equation_tech" target="_blank">Plac Equation</a><br />
</div><br />
<br />
<div style="display:inline-block;text-align:center;float:right"><br />
So the equations for the promoters are these:<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/8/87/3%29_equations_promoters.png" width=430px/><br/><br />
Figure 7: Promoters' equations<br><br />
</div><br />
<div style="clear:both"></div><br />
<br/><br />
<div class="petitSsTitre">Inducers</div><br />
With the same method based on law of mass action and binding/unbinding kinetic, we obtain the inducers' equations.<br/><br/><br />
<br />
<div style="display:inline-block;text-align:center"><br />
<iframe frameborder="0" width="430" height="250" src="http://www.dailymotion.com/embed/video/xttbsf"></iframe><br /><a href="http://www.dailymotion.com/video/xttbsf_inducer-equation_tech" target="_blank">Inducer Equation</a><br />
</div><br />
<br />
<div style="display:inline-block;text-align:center;float:right;margin-top:30px"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/3%29_equations_inducers.png" width=430px/><br/><br />
Figure 8: Inducers' equations<br/><br />
</div><br />
<div style="clear:both"></div><br />
<br><br />
<div class="petitSsTitre">Repressors</div><br />
Now, for the repressors, the method is quite similar as before. However, we have to take into account that the proteins have a degradation rate (&delta;) depending on their nature and the environment. The quantity of protein produced at each time depends on the promoter under control (&alpha;). <br><br><br />
<br />
<center><br />
<iframe frameborder="0" width="480" height="270" src="http://www.dailymotion.com/embed/video/xtuc6t?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtuc6t_lacinequation_tech" target="_blank">LacI Equation</a> </center><br />
<br />
<br><br><br />
We obtain XylR's equation exactly as LacI's. <br><br> <br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c8/3%29_LacI_and_XylR_equations.png" width = 90%/></center><br />
<center>Figure 9: Repressors' equations <br></center><br><br><br />
<br />
<br />
<h3>Parameters of the model</h3><br />
<br><br />
For this model, we need at least 12 parameters that characterize the variables and their relationship between each other. <br />
The available values have been measure mainly in an <i>E. coli</i> strain. <br />
<br><br><br />
<center><br />
<table><br />
<tr><br />
<th>Name</th><br />
<th>Description</th><br />
<th>Unit</th><br />
<th>Value</th><br />
<th>Reference</th><br />
</tr><br />
<br />
<tr><br />
<td>Prod_Plac</td><br />
<td>Production rate from <i>Plac</i> promoter</td><br />
<td>mol.s-1</td><br />
<td>1.66E23</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Prod_Pxyl</td><br />
<td>Production rate from <i>Pxyl</i> promoter</td><br />
<td>mol.s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
<tr><br />
<td>k1</td><br />
<td>binding kinetic of LacI and IPTG</td><br />
<td>mol-1.s-1</td><br />
<td>1.2E5</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>km1</td><br />
<td>unbinding kinetic of LacI_IPTG</td><br />
<td>s-1</td><br />
<td>2.1E-1</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>k2</td><br />
<td>binding kinetic of XylR and Xylose</td><br />
<td>mol-1.s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
<tr><br />
<td>km2</td><br />
<td>unbinding kinetic of XylR_Xylose</td><br />
<td>s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
<tr><br />
<td>k3</td><br />
<td>binding kinetic of LacI and <i>Plac</i></td><br />
<td>mol-1.s-1</td><br />
<td>5.1E6</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>km3</td><br />
<td>unbinding kinetic of <i>PlacO</i></td><br />
<td>s-1</td><br />
<td>3.7E-2</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>k4</td><br />
<td>binding kinetic of XylR and <i>Plac</i></td><br />
<td>mol-1.s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
<tr><br />
<td>km4</td><br />
<td>unbinding kinetic of XylR and <i>PlacO</i></td><br />
<td>s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
<tr><br />
<td>&delta;_LacI</td><br />
<td>degradation rate of LacI</td><br />
<td>s-1</td><br />
<td></td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>&delta;_XylR</td><br />
<td>degradation rate of XylR</td><br />
<td>s-1</td><br />
<td>***</td><br />
<td>**</td><br />
</tr><br />
</table><br><br />
Model parameters. <small>*** for no value and ** for no reference</small><br />
</center> <br />
<br />
<br><br><br />
<h3>Hypotheses</h3><br />
<br><br />
The following hypotheses have been made for this model. <br />
<ul><br />
<li>we just need LacI concentration for surfactant production and not Sfp and AbrB concentration because there is a proportional link between them. If there are LacI proteins produced, there will be also Sfp and AbrB proteins.</li><br />
<li>we assumed that there will be no degradation of IPTG due to its high stability[4] and no metabolism of Xylose in our condition[5].</li><br />
<li>we are aware of LacI[6] and XylR[7] dimerisation as fundamental functional unit but they are not taken into account in this model.</li><br />
</ul><br />
<br><br />
<br />
<div style="font-size:12px"><br />
<b>References:</b><br><br />
<ul><br />
<li> [1] Nature. 2000 Jan 20;403(6767):335-8.<i> A synthetic oscillatory network of transcriptional regulators.</i> Elowitz MB, Leibler S. <br />
<li> [2] Xu H.,Moraitis M., Reedstrom R. J., Matthews K. S. 1998. <i>Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA.</i> J. Biol. Chem. 273:8958–8964.<br />
<li> [3] Tuttle et al. <i>Model-Driven Designs of an Oscillating Gene Network.</i>, Biophys J 89(6):3873-3883, 2005<br />
<li> [4] Herzenberg, L.A., <i>Studies on the induction of beta-galactosidase in a cryptic strain of Escherichia coli.</i> Biochim. Biophys. Acta, 31, 525 (1959)<br />
<li> [5] http://bsubcyc.org/BSUB/NEW-IMAGE?type=PATHWAY&object=XYLCAT-PWY<br />
<li> [6] Ramot, R. et al, <i>Lactose Repressor Experimental Folding Landscape: Fundamental Functional Unit and Tetramer Folding Mechanisms</i>. Biochemistry (2012)<br />
<li> [7] Song S., Park C. <i>Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator.</i> J Bacteriol. 1997 Nov;179(22):7025-32.<br />
</ul><br />
</div><br />
<br />
</div><br />
</div><br />
<h2>Results</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<br><br />
<div class="petitSsTitre">Expected results</div><br />
Finally, we translated the biological system into mathematical equations. As we can see, there is a lot of paramaters for the binding and unbinding kinetics, degradation rates and productions from promoters. However, values for only a few of them are available. This is why we needed to simulate the behaviour of our system. <br><br />
We want to have an overproduction of XylR in the presence of IPTG, for the STICK option. And an overproduction of LacI when there is xylose in the environment, for the COAT option. <br><br />
<br />
According to the above theoretical model, we should generate the expected toggle switch (figure below), using the appropriate values for all parameters.<br><br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/e/e6/4%29_COAT.png" width=300px/><br />
<img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/5/51/4%29_Stick.png" width=300px/></center><br><br />
<center>Figure 10: Expected results of the model</center><br><br />
<br />
<br />
<br />
<br />
<div class="petitSsTitre">Experimentations</div><br />
So far, we can consider the values for the P<sub><i>lac</i></sub> promoter, LacI and IPTG that have been measured in <i>E. coli</i> to apply to our <i>B. subtilis</i> model. We have performed experiments with the P<sub><i>xyl</i></sub>, xylose and XylR to evaluate whether this promoter can be modelled using similar values. When this step is performed, our model will allow us to determine two important concentration limits: <br><br />
<ul><br />
<li>1) the lowest IPTG concentration for the induction of the COAT option;</li><br />
<li>2) the lowest xylose concentration for the induction of the STICK option.</li><br />
</ul><br />
<br><br />
<br />
<br><br><br />
We also thought of a way to obtain binding and unbinding kinetics of the proteins. Some methods such as Isothermal titration calorimetry (ITC) permit to determine the thermodynamic parameters of interactions in solution<br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
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<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSATeam:Lyon-INSA2012-10-26T22:56:26Z<p>Suxiaohui: </p>
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</center><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png"/><br />
</center><br />
<div ><h1>Team project description</h1></div><br />
<br/><br />
<div style="margin:20px;margin-top:0;font-size:14px;"><br />
Biofilms are responsible for billions of dollars in production losses and treatment costs in the industry every year. Biofilm-related problems are major concerns in the food industry where it can cause food spoilage or poisoning, in health industry because of pathogens' persistence and dispersal, or in the oil and water industry where it causes corrosion. Assuming that the environment is already over-saturated with harmful chemical products such as biocides, whose long term health effects remain to be elucidated, <b>there is a great need for novel solutions to reduce detrimental biofilm effects</b>.<br/><br />
<br/><br />
<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtsdia?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtsdia_our-project-presentation-biofilm-killer-lyon-insa-igem_tech" target="_blank"><font color ="purple">Our project presentation Biofilm Killer Lyon...</font></a></center><br><br />
<br />
<br />
<b>To reduce the use of biocides</b>, the Lyon-INSA iGEM team aims to <b>engineer bacterial "torpedo" capable to infiltrate and destroy biofilms</b> formed on industrial equipments, pipes or reservoirs. Industrial surfaces will then be protected from further deleterious contamination by either a <b>surfactant coating</b>, or the <b>establishment of a protective biofilm</b>.<br/><br />
<br/><br />
<br />
Our experimental model consists of <b><i>Staphylococcus epidermidis</i> as the detrimental biofilm</b>, and <b><i>Bacillus subtilis</i> as the "Biofilm Killer" agent</b>. Three complementary modules will be constructed to arm our "Biofilm Killer" strain:<br/><br />
<br/><br />
<div class=indented2 style="margin:20px;"><br />
<li>The first step will be to fit <i>Bacillus subtilis</i> swimmers with both a <b>biocide and a scattering agent</b>. Irrigation of these active substances in the biofilm should be facilitated by the tunneling activity of these swimmers cells.</li><br />
<li>Then, to protect the surface which used to be the biofilm's substrate from next possible bacterial adhesion, we will engineered the <b>conditional production of surfactin</b>, a naturally toxic bio-surfactant produced by <i>B. subtilis</i> and displaying well-known antimicrobial properties.</li><br />
<li>Finally, the <b>conditional establishment of a competitive barrier flora</b>, constituted of <i>B. subtilis</i> in our model, will be achieved by inhibiting the main biofilm repressor abrB gene. </li><br/> <br />
</div><br />
<br />
<br />
<i>Bacillus</i> strains are non-pathogenic, and do not cause equipment degradation by corrosion: their settlement on surfaces represents a biocide-alternative strategy to prevent the formation of new dangerous biofilm. This project provides a potential economy, and environmental friendly solution for the control of unwanted biofilm. <br />
<br />
<br />
<br/><br />
</div><br />
</div><br />
</div><br />
<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSATeam:Lyon-INSA2012-10-26T22:53:29Z<p>Suxiaohui: </p>
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<center><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png"/><br />
</center><br />
<div ><h1>Team project description</h1></div><br />
<br/><br />
<div style="margin:20px;margin-top:0;font-size:14px;"><br />
Biofilms are responsible for billions of dollars in production losses and treatment costs in the industry every year. Biofilm-related problems are major concerns in the food industry where it can cause food spoilage or poisoning, in health industry because of pathogens' persistence and dispersal, or in the oil and water industry where it causes corrosion. Assuming that the environment is already over-saturated with harmful chemical products such as biocides, whose long term health effects remain to be elucidated, <b>there is a great need for novel solutions to reduce detrimental biofilm effects</b>.<br/><br />
<br/><br />
<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtsdia?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtsdia_our-project-presentation-biofilm-killer-lyon-insa-igem_tech" target="_blank"><font color ="purple">Our project presentation Biofilm Killer Lyon...</font></a></center><br><br />
<br />
<br />
<b>To reduce the use of biocides</b>, the Lyon-INSA iGEM team aims at <b>engineering bacterial "torpedo" capable to infiltrate and destroy biofilms</b> formed on industrial equipments, pipes or reservoirs. Industrial surfaces will then be protected from further deleterious contamination by either a <b>surfactant coating</b>, or the <b>establishment of a protective biofilm</b>.<br/><br />
<br/><br />
<br />
Our experimental model consists of <b><i>Staphylococcus epidermidis</i> as the detrimental biofilm</b>, and <b><i>Bacillus subtilis</i> as the "Biofilm Killer" agent</b>. Three complementary modules will be constructed to arm our "Biofilm Killer" strain:<br/><br />
<br/><br />
<div class=indented2 style="margin:20px;"><br />
<li>The first step will be to fit <i>Bacillus subtilis</i> swimmers with both a <b>biocide and a scattering agent</b>. Irrigation of these active substances in the biofilm should be facilitated by the tunneling activity of these swimmers cells.</li><br />
<li>Then, to protect the surface which used to be the biofilm's substrate from next possible bacterial adhesion, we will engineered the <b>conditional production of surfactin</b>, a naturally toxic bio-surfactant produced by <i>B. subtilis</i> and displaying well-known antimicrobial properties.</li><br />
<li>Finally, the <b>conditional establishment of a competitive barrier flora</b>, constituted of <i>B. subtilis</i> in our model, will be achieved by inhibiting the main biofilm repressor abrB gene. </li><br/> <br />
</div><br />
<br />
<br />
<i>Bacillus</i> strains are non-pathogenic, and do not cause equipment degradation by corrosion: their settlement on surfaces represents a biocide-alternative strategy to prevent the formation of new dangerous biofilm. This project provides a potential economy, and environmental friendly solution for the control of unwanted biofilm. <br />
<br />
<br />
<br/><br />
</div><br />
</div><br />
</div><br />
<br />
</body><br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/TeamTeam:Lyon-INSA/Team2012-10-26T01:35:20Z<p>Suxiaohui: </p>
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<h1>Our Team</h1><br />
<br />
<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br />
<br />
<h2>Students</h2><br />
<div class="wrapper"><br />
<br />
<div id="studentListing"><br />
<div class="studentName"><br />
Alex Mizgier<br />
</div><br />
<div class="studentName"><br />
Alexandre Duprey<br />
</div><br />
<div class="studentName"><br />
Anne Haziza<br />
</div><br />
<div class="studentName"><br />
Audrey Masi<br />
</div> <br />
<div class="studentName"><br />
Bastien Doix<br />
</div><br />
<div class="studentName"><br />
Béryl Royer-Bertrand<br />
</div><br />
<div class="studentName"><br />
Carine Gimbert<br />
</div><br />
<div class="studentName"><br />
Clémence Gonthier<br />
</div><br />
<div class="studentName"><br />
Ioana Sandu<br />
</div><br />
<div class="studentName"><br />
Marion Traouan<br />
</div><br />
<div class="studentName"><br />
Marion Wolfovski<br />
</div><br />
<div class="studentName"><br />
Patricia Gifu<br />
</div> <br />
<div class="studentName"><br />
Rémi Hocq<br />
</div><br />
<div class="studentName"><br />
Viviane Chansavang<br />
</div> <br />
<div class="studentName"><br />
Xavier Tholot<br />
</div><br />
</div><br />
<br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alex Mizgier<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2f/Tetealexser.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Hairix, I am a third-year biochemist at INSA Lyon. I am from Chile, specifically from the northern limit of Western Patagonia.<br/><br />
I got into the iGEM project because I think that itís an unique experience and an excellent opportunity to learn more about bacterial manipulation and synthetic biology.<br/><br />
Apart from microbiology, I like reading, playing rugby, bicycle touring, and everything that involves nature and outdoor activities. You will find in me a knight-errant.<br/></p><br />
</div><br />
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<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alexandre Duprey<br />
</div><br />
<div class="photoPres"><br />
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<div class="textePres"><br />
<p>This year, I'm back for another iGEM competition. I'm still at INSA Lyon, fourth-year biochemistry and biotechnology, and still interested in how we can regulate parts to make them work together. However this time I'm rather advising on the project with the intent to improve our weak points (who said modelling ?), and making sure the newcomers donít make (too many) mistakes. </br> Otherwise I play video games. A lot. Too much...</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Anne Haziza<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p> I am a third-year student in Biochemistry and Biotechnologies at INSA Lyon. I am taking part in the iGEM competition for the first time. This project is a good way for me to improve my organization skills, ingenuity and dynamism which will be very useful for the future. </br>Outside of school, I enjoy cooking, playing handball and mainly traveling whenever possible! So I believe on the talent of her team to discover Amsterdam and of course Boston! </p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Audrey Masi<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4f/Audre.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/e/e4/Audrey_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Squirrelix because I used to climb up on trees and hide in narrow cupboards to make jokes to my friends.<br><br />
I am a third-year bioengineer and I am joining the iGEM team after a first experience in molecular biology to improve my knowledge and discover the synthetic biology approach.<br><br />
Besides iGEM, I am interested in rock dancing, cooking, traveling, and above all that literature.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Bastien Doix<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year student in Biochemistry and Biotechnology at INSA Lyon and taking part in iGEM for the first time. I am joining the iGEM team to have a first sight of what genetics and bacterial manipulation is and to step into something different from studies.</br> I like sports, skiing and computer graphics and hope to make it to Boston.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Béryl Royer-Bertrand<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
</div><br />
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<div class="textePres"><br />
<p>This is my second participation in the iGEM competition with the INSA Lyon team. In fourth year in Bioinformatics and Modeling, Iím helping this year the team in the modelling part from Scotland, where I am in academic exchange. </p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Carine Gimbert<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
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<div class="textePres"><br />
<p>Also known as Stoatix, it is my first participation in the INSA-Lyon team. In my previous studies, I worked on two projects about bioremediation and production of vanillin using biotechnology. So being part of the iGEM team is really important to continue learning how to manage bacteria and working in a student team. </br>I am fond of dance, photography and art in general. I live in a town where the international short film festival takes place : Clermont-Ferrand and one of my favorite is <a href="http://www.youtube.com/watch?v=DdLehwjV4pc">La révolution des crabesî</a></p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Clémence Gonthier<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>When you participate in iGEM once, you just want to take part in this great adventure again !<br><br />
In my fourth year of Biochemistry and Biotechnology, I'm still enjoying microbiology, modifying bacteria and creating new plasmids!<br><br />
I am this time on the advisor side... Helping the new team members searching for sponsors and guiding them for experiments in the lab.<br><br />
In my free time, I like dancing, playing the piano and traveling !</p><br />
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Ioana Sandu<br />
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<p>I am a third-year Biochemistry student. I decided to join the Lyon team because I believe that the iGEM competition is a challenging experience that will put myself to the test. </br>Apart from synthetic biology, I like dancing, star-gazing and reading while listening to music (the older, the better).</p><br />
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Marion Traouan<br />
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<p>I am an undergraduate student in biochemistry and biotechnologies at INSA Lyon. This is my first participation in an iGEM team. I consider this as an opportunity not only to acquire specific qualifications very useful for the future, but also to participate in the construction of an attractive project from scratch.<br/><br />
Besides my interest in biosciences, I am into sports like basket-ball or volley-ball, reading and cinema. I hope my "blondness" won't be an obstacle to the realization of this project.</p><br />
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Marion Wolfovski<br />
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<p>Also known as Rasberrix Olympix, I have a bachelor's degree in Sciences and I am now studying Biochemistry and Biotechnologies. This is my first participation in the iGEM competition. I love new challenges and iGEM is an amazing opportunity to manage together my passion for biology and building a project : a scientific adventure.</br> <br />
I love sports and making cookies for my lovely team !</p><br />
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Patricia Gifu<br />
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<p>I am an undergraduate student in Biochemistry and Biotechnology at INSA de Lyon. This year, I participate for the first time in iGEM. I am very confident that INSA Lyon team is going to win because the team members worked very hard on the project and they all are skillful. I am passionate by biosciences. </br>Outside the school I like watching movies and traveling whenever I have time.</p><br />
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Rémi Hocq<br />
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<img src="https://static.igem.org/mediawiki/2012/thumb/8/8c/Remy.jpg/774px-Remy.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/thumb/1/10/Remy2.JPG/397px-Remy2.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am also known as Clumsix for often being Ö clumsy. Hell, I am the kind of guy who managed to cut my finger trying to open a bottle of beer and for the record, I was actually using a bottle-opener. My clumsiness is even contagious : GaÎl and Yoann also cut their finger during the summer.</br><br />
Anyway, I am also a third-year bioengineering student who love music (especially hard rock !), good food (though I am skinny and absolutely don't know why) and traveling. I may be a bit of a nerd too as I am a videogames fanatic.</p><br />
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Viviane Chansavang<br />
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<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/a/a0/VivianeFun.png" width="180px"/><br />
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<div class="textePres"><br />
<p>iGEM rocks so badly, when you do it once, you just wanna do it again :) <br><br> <br />
But this year, I'm keeping a low profile, just helping out the newbies to settle in and feel comfy in the lab. I'm still enjoying working around bacteria, apart from <i>B. subtilis</i> producing lysostaphin, because it smells so badly you just wanna die when you happen to be working in the same room!<br><br><br />
I also like playing volleyball, cooking, baking, eating and above all traveling and meeting new people ^^ See y'all at the Jamboree! </p><br />
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Xavier Tholot<br />
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<p>I am a third-year student in computer sciences at INSA Lyon. <br/><br />
I come from Clermont-Ferrand, the city where Michelin was created, which isn't far from Lyon.<br/><br />
I am fond of music (I play the guitar and the clarinet), and also of computers : this year, I joined the Lyon-INSA team to help them improving their wiki, and make it as great as possible.</p> <br />
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<h2>Advisors</h2><br />
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Fanny Springer<br />
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Gaël Chambonnier<br />
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Philippe Thomas<br />
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Romain Briandet<br />
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Fanny Springer<br />
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<p>I've joined the Lyon-INSA team this year to see how we can change the world using genetically engineered material !</p><br />
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Gaël Chambonnier<br />
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<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Hi everybody, here is Chambix!! I just graduated from the INSA Lyon in Biotechnologies but after two participations at the iGEM Competition, I couldnít do anything but continue my studies with a master in bioinformatics.<br><br />
After two years working in the lab for the team, I came back this year trying to help out the new team, sharing my experiences. Actually, they didnít really need my help. Theyíve done a great job with Bacillix. And for sure you'll have to deal with them in Amsterdam and Boston.</p><br />
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Philippe Thomas<br />
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<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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<p>This year, Asterix and his friends have travelled until Buenos Aires in Argentina... They meet some strange people living riding horses, drinking mate, eating very good meat and raising cows in the infinite pampa! I'm one of this gauchos! I'm currently working in Buenos Aires for 18 months as an Engineer in Biotechnologies at Sanofi Pasteur. As it is not so easy to assist meeting and perform some experiments, the best for me was to be an advisor!</br><br />
I try to help out my Gallic friends when some extra work is needed and give my best as advisor, sharing my experiences of the iGEM competition with them! El hombre de la pampa can't wait to see the Lyon Biosciences Team rockiní Amsterdam and Boston with our very powerful bacteria! </p><br />
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Romain Briandet<br />
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<p>I am the leader of the biofilm group at the INRA Micalis Institute. I have focused my research on microbial biofilms present in the food chain with special emphasis on their role in the persistence of pathogens. One of my main scientific line of interest is to identify the link between the 3D spatial organization of biofilms and the survival mechanisms of cells in face to the exposition of antimicrobials. Recently, my team reported that a tiny proportion of certain <i>Bacillus</i> species can tunnel through biofilms, creating pores that allow molecules to flow in. We are now evaluating the industrial benefit to pretreat target biofilms with swimming <i>Bacilli</i> cocktails to increase their efficacy and reduced their ecological impact.</p><br />
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<h2>Instructors</h2><br />
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Agnès Rodrigue<br />
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Corinne Dorel<br />
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Olivier Brette<br />
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Philippe Oger<br />
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Valérie Desjardin<br />
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Yoann Louis<br />
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Agnès Rodrigue<br />
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<p>As an associate professor I find that iGEM is an unique experience, a place to mix teaching and practice in a long term project relying on studentsí motivation. My teaching interests are microbiology, molecular biology, protein engineering and bioinformatics. <br/> My research topic focus on understanding the molecular mechanisms involved in bacterial adaptation to a metal-rich environment, from molecular interactions to population biology. Apart from the basic approaches, this subject also offers opportunities to develop biological tools for bioremediation of spoiled environment or <i>in situ</i> detection of toxic compounds for instance, developments which I'm interested in. My motivation for iGEM is the same : conception and application. </p><br />
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<div class="presentation insPres"><br />
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Corinne Dorel<br />
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<p>My teaching activities in the Microbial Genetics mainly take place within the Biosciences department at INSA Lyon, but also at the Ecole Normale Supérieur de Lyon and at the University Claude Bernard Lyon 1.<br/><br/><br />
My research work is based on the understanding of genetic mechanisms involved in the formation of biofilms and the contamination of materials. Being the Communications officer for the Biosciences department, my participation in iGEM 2012 is part of a strategy to promote and share knowledge in the field of research in genetic engineering and more generally in Biological Sciences.</p><br />
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<div class="presentation insPres"><br />
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Olivier Brette<br />
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<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
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<p>Associate Professor in economics at the Institut National des Sciences Appliquées (INSA) de Lyon</p><br />
<p>I teach the "economics of firm", "innovation economics", as well as the "economics of globalization" to engineering students.</p><br />
<p>I am affiliated with the CNRS Research Unit "Environment, City, Society" (EVS), where I pursue my research activities in theoretical and applied economics. From a theoretical viewpoint, my research work aims at developing the methodological, conceptual, as well as behavioral foundations of Institutional and Evolutionary Economics. I resort to this theoretical framework to deal with different kinds of issues in innovation economics and economic geography.</p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Philippe Oger<br />
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<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
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<p>Also known as Piezophilix because I work on high-pressure adaptation in microorganisms.<br />
My research is focussed on adaptation mechanisms in microbes from the deep-biosphere, and mainly in Archaea isolated from deep-sea hydrothermal vent systems.<br><br><br />
Our aim is to identify and quantify what genetic modifications make our favorite model, <i>Pyrococcus yanaosii</i>, require 500 times the atmospheric pressure for growth, when everybody else's favorite lab rat, <i>E. coli</i>, cannot even growth at the same hydrostatic pressure.<br><br><br />
My teaching activities at the University of Lyon deal with petroleum reservoir microbiology and the use of biosignatures for the study of past and present environments.</p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Valérie Desjardin<br />
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<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
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<p>Associate professor at the Institut National des Sciences Appliquées de Lyon</p><br />
<p>After a Master of Advanced Studies in Biochemistry and a PhD in Chemistry and Science and Techniques of waste, now I teach Chemistry classes and also a class dealing with radioactive waste management in the Energy Engineering and Environment department. <br/> My research work at the Laboratory of Civil engineering and Environmental engineering (LGCIE) is currently aimed at the study of biophysicochemical interactions of pollutants in various compounds (soils, sediments, municipal solid waste) using molecular biology tools. I am very excited to take part in the iGEM 2012 project which leads to the development of a synergy, already launched, between the Biosciences department and Environmental sciences.</p><br />
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<div class="presentation insPres"><br />
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Yoann Louis<br />
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<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
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<p>Young ;) ) Associate professor at the Institut National des Sciences Appliquées de Lyon</p><br />
<p>Up to now my work is focused on the trace metal behavior in the environment and their interaction with dissolved organic matter in aquatic ecosystems.</p><br />
<p>Now my work at the LGCIE laboratory is mainly <strike>to develop a magic potion to be stronger than Asterix </strike>to study trace metals/ organic matter behavior in various waste and to give an expertise on their potential toxicity depending on the bio-physico-chemical conditions of the studied site.<br />
As a chemist, my interest in the iGEM project is to have a working approach angle enabling improvements of our environment thanks to our complementarity.</p><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/File:VivianeFun.pngFile:VivianeFun.png2012-10-26T01:31:36Z<p>Suxiaohui: uploaded a new version of &quot;File:VivianeFun.png&quot;</p>
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<div></div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/TeamTeam:Lyon-INSA/Team2012-10-26T01:30:01Z<p>Suxiaohui: </p>
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<h1>Our Team</h1><br />
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<h2>Students</h2><br />
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Alex Mizgier<br />
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Alexandre Duprey<br />
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Anne Haziza<br />
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Audrey Masi<br />
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Bastien Doix<br />
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Béryl Royer-Bertrand<br />
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Carine Gimbert<br />
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Clémence Gonthier<br />
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Ioana Sandu<br />
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Marion Traouan<br />
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Marion Wolfovski<br />
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Patricia Gifu<br />
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Rémi Hocq<br />
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Viviane Chansavang<br />
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Xavier Tholot<br />
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Alex Mizgier<br />
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<p>Also known as Hairix, I am a third-year biochemist at INSA Lyon. I am from Chile, specifically from the northern limit of Western Patagonia.<br/><br />
I got into the iGEM project because I think that itís an unique experience and an excellent opportunity to learn more about bacterial manipulation and synthetic biology.<br/><br />
Apart from microbiology, I like reading, playing rugby, bicycle touring, and everything that involves nature and outdoor activities. You will find in me a knight-errant.<br/></p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alexandre Duprey<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/AlexD.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/6/64/Alexmizgier.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>This year, I'm back for another iGEM competition. I'm still at INSA Lyon, fourth-year biochemistry and biotechnology, and still interested in how we can regulate parts to make them work together. However this time I'm rather advising on the project with the intent to improve our weak points (who said modelling ?), and making sure the newcomers donít make (too many) mistakes. </br> Otherwise I play video games. A lot. Too much...</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Anne Haziza<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p> I am a third-year student in Biochemistry and Biotechnologies at INSA Lyon. I am taking part in the iGEM competition for the first time. This project is a good way for me to improve my organization skills, ingenuity and dynamism which will be very useful for the future. </br>Outside of school, I enjoy cooking, playing handball and mainly traveling whenever possible! So I believe on the talent of her team to discover Amsterdam and of course Boston! </p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Audrey Masi<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4f/Audre.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Audrey_fun.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Also known as Squirrelix because I used to climb up on trees and hide in narrow cupboards to make jokes to my friends.<br><br />
I am a third-year bioengineer and I am joining the iGEM team after a first experience in molecular biology to improve my knowledge and discover the synthetic biology approach.<br><br />
Besides iGEM, I am interested in rock dancing, cooking, traveling, and above all that literature.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Bastien Doix<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am a third-year student in Biochemistry and Biotechnology at INSA Lyon and taking part in iGEM for the first time. I am joining the iGEM team to have a first sight of what genetics and bacterial manipulation is and to step into something different from studies.</br> I like sports, skiing and computer graphics and hope to make it to Boston.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Béryl Royer-Bertrand<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>This is my second participation in the iGEM competition with the INSA Lyon team. In fourth year in Bioinformatics and Modeling, Iím helping this year the team in the modelling part from Scotland, where I am in academic exchange. </p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Carine Gimbert<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/dc/Carine.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Carine_fun.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Also known as Stoatix, it is my first participation in the INSA-Lyon team. In my previous studies, I worked on two projects about bioremediation and production of vanillin using biotechnology. So being part of the iGEM team is really important to continue learning how to manage bacteria and working in a student team. </br>I am fond of dance, photography and art in general. I live in a town where the international short film festival takes place : Clermont-Ferrand and one of my favorite is <a href="http://www.youtube.com/watch?v=DdLehwjV4pc">La révolution des crabesî</a></p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Clémence Gonthier<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>When you participate in iGEM once, you just want to take part in this great adventure again !<br><br />
In my fourth year of Biochemistry and Biotechnology, I'm still enjoying microbiology, modifying bacteria and creating new plasmids!<br><br />
I am this time on the advisor side... Helping the new team members searching for sponsors and guiding them for experiments in the lab.<br><br />
In my free time, I like dancing, playing the piano and traveling !</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Ioana Sandu<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am a third-year Biochemistry student. I decided to join the Lyon team because I believe that the iGEM competition is a challenging experience that will put myself to the test. </br>Apart from synthetic biology, I like dancing, star-gazing and reading while listening to music (the older, the better).</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Traouan<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am an undergraduate student in biochemistry and biotechnologies at INSA Lyon. This is my first participation in an iGEM team. I consider this as an opportunity not only to acquire specific qualifications very useful for the future, but also to participate in the construction of an attractive project from scratch.<br/><br />
Besides my interest in biosciences, I am into sports like basket-ball or volley-ball, reading and cinema. I hope my "blondness" won't be an obstacle to the realization of this project.</p><br />
</div><br />
</div> <br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Wolfovski<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/58/MarionW.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8b/MarionW_fun.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Also known as Rasberrix Olympix, I have a bachelor's degree in Sciences and I am now studying Biochemistry and Biotechnologies. This is my first participation in the iGEM competition. I love new challenges and iGEM is an amazing opportunity to manage together my passion for biology and building a project : a scientific adventure.</br> <br />
I love sports and making cookies for my lovely team !</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Patricia Gifu<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am an undergraduate student in Biochemistry and Biotechnology at INSA de Lyon. This year, I participate for the first time in iGEM. I am very confident that INSA Lyon team is going to win because the team members worked very hard on the project and they all are skillful. I am passionate by biosciences. </br>Outside the school I like watching movies and traveling whenever I have time.</p><br />
</div> <br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Rémi Hocq<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8c/Remy.jpg/774px-Remy.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/1/10/Remy2.JPG/397px-Remy2.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am also known as Clumsix for often being Ö clumsy. Hell, I am the kind of guy who managed to cut my finger trying to open a bottle of beer and for the record, I was actually using a bottle-opener. My clumsiness is even contagious : GaÎl and Yoann also cut their finger during the summer.</br><br />
Anyway, I am also a third-year bioengineering student who love music (especially hard rock !), good food (though I am skinny and absolutely don't know why) and traveling. I may be a bit of a nerd too as I am a videogames fanatic.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Viviane Chansavang<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a0/VivianeFun.png" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>iGEM rocks so badly, when you do it once, you just wanna do it again :) <br><br> <br />
But this year, I'm keeping a low profile, just helping out the newbies to settle in and feel comfy in the lab. I'm still enjoying working around bacteria, apart from <i>B. subtilis</i> producing lysostaphin, because it smells so badly you just wanna die when you happen to be working in the same room!<br><br><br />
I also like playing volleyball, cooking, baking, eating and above all traveling and meeting new people ^^ See y'all at the Jamboree! </p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Xavier Tholot<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am a third-year student in computer sciences at INSA Lyon. <br/><br />
I come from Clermont-Ferrand, the city where Michelin was created, which isn't far from Lyon.<br/><br />
I am fond of music (I play the guitar and the clarinet), and also of computers : this year, I joined the Lyon-INSA team to help them improving their wiki, and make it as great as possible.</p> <br />
</div><br />
</div><br />
</div><br />
<br />
<h2>Advisors</h2><br />
<div class="wrapper"><br />
<div id="advisorListing"><br />
<div class="advisorName"><br />
Fanny Springer<br />
</div><br />
<div class="advisorName"><br />
Gaël Chambonnier<br />
</div><br />
<div class="advisorName"><br />
Philippe Thomas<br />
</div><br />
<div class="advisorName"><br />
Romain Briandet<br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Fanny Springer<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I've joined the Lyon-INSA team this year to see how we can change the world using genetically engineered material !</p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Gaël Chambonnier<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
</div> <br />
</div><br />
<div class="textePres"><br />
<p>Hi everybody, here is Chambix!! I just graduated from the INSA Lyon in Biotechnologies but after two participations at the iGEM Competition, I couldnít do anything but continue my studies with a master in bioinformatics.<br><br />
After two years working in the lab for the team, I came back this year trying to help out the new team, sharing my experiences. Actually, they didnít really need my help. Theyíve done a great job with Bacillix. And for sure you'll have to deal with them in Amsterdam and Boston.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Philippe Thomas<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>This year, Asterix and his friends have travelled until Buenos Aires in Argentina... They meet some strange people living riding horses, drinking mate, eating very good meat and raising cows in the infinite pampa! I'm one of this gauchos! I'm currently working in Buenos Aires for 18 months as an Engineer in Biotechnologies at Sanofi Pasteur. As it is not so easy to assist meeting and perform some experiments, the best for me was to be an advisor!</br><br />
I try to help out my Gallic friends when some extra work is needed and give my best as advisor, sharing my experiences of the iGEM competition with them! El hombre de la pampa can't wait to see the Lyon Biosciences Team rockiní Amsterdam and Boston with our very powerful bacteria! </p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Romain Briandet<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am the leader of the biofilm group at the INRA Micalis Institute. I have focused my research on microbial biofilms present in the food chain with special emphasis on their role in the persistence of pathogens. One of my main scientific line of interest is to identify the link between the 3D spatial organization of biofilms and the survival mechanisms of cells in face to the exposition of antimicrobials. Recently, my team reported that a tiny proportion of certain <i>Bacillus</i> species can tunnel through biofilms, creating pores that allow molecules to flow in. We are now evaluating the industrial benefit to pretreat target biofilms with swimming <i>Bacilli</i> cocktails to increase their efficacy and reduced their ecological impact.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<h2>Instructors</h2><br />
<div class="wrapper"><br />
<div id="instructorListing"><br />
<div class="instructorName"><br />
Agnès Rodrigue<br />
</div><br />
<div class="instructorName"><br />
Corinne Dorel<br />
</div><br />
<div class="instructorName"><br />
Olivier Brette<br />
</div><br />
<div class="instructorName"><br />
Philippe Oger<br />
</div><br />
<div class="instructorName"><br />
Valérie Desjardin<br />
</div><br />
<div class="instructorName"><br />
Yoann Louis<br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Agnès Rodrigue<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>As an associate professor I find that iGEM is an unique experience, a place to mix teaching and practice in a long term project relying on studentsí motivation. My teaching interests are microbiology, molecular biology, protein engineering and bioinformatics. <br/> My research topic focus on understanding the molecular mechanisms involved in bacterial adaptation to a metal-rich environment, from molecular interactions to population biology. Apart from the basic approaches, this subject also offers opportunities to develop biological tools for bioremediation of spoiled environment or <i>in situ</i> detection of toxic compounds for instance, developments which I'm interested in. My motivation for iGEM is the same : conception and application. </p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Corinne Dorel<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>My teaching activities in the Microbial Genetics mainly take place within the Biosciences department at INSA Lyon, but also at the Ecole Normale Supérieur de Lyon and at the University Claude Bernard Lyon 1.<br/><br/><br />
My research work is based on the understanding of genetic mechanisms involved in the formation of biofilms and the contamination of materials. Being the Communications officer for the Biosciences department, my participation in iGEM 2012 is part of a strategy to promote and share knowledge in the field of research in genetic engineering and more generally in Biological Sciences.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Olivier Brette<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Associate Professor in economics at the Institut National des Sciences Appliquées (INSA) de Lyon</p><br />
<p>I teach the "economics of firm", "innovation economics", as well as the "economics of globalization" to engineering students.</p><br />
<p>I am affiliated with the CNRS Research Unit "Environment, City, Society" (EVS), where I pursue my research activities in theoretical and applied economics. From a theoretical viewpoint, my research work aims at developing the methodological, conceptual, as well as behavioral foundations of Institutional and Evolutionary Economics. I resort to this theoretical framework to deal with different kinds of issues in innovation economics and economic geography.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Philippe Oger<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Also known as Piezophilix because I work on high-pressure adaptation in microorganisms.<br />
My research is focussed on adaptation mechanisms in microbes from the deep-biosphere, and mainly in Archaea isolated from deep-sea hydrothermal vent systems.<br><br><br />
Our aim is to identify and quantify what genetic modifications make our favorite model, <i>Pyrococcus yanaosii</i>, require 500 times the atmospheric pressure for growth, when everybody else's favorite lab rat, <i>E. coli</i>, cannot even growth at the same hydrostatic pressure.<br><br><br />
My teaching activities at the University of Lyon deal with petroleum reservoir microbiology and the use of biosignatures for the study of past and present environments.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Valérie Desjardin<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Associate professor at the Institut National des Sciences Appliquées de Lyon</p><br />
<p>After a Master of Advanced Studies in Biochemistry and a PhD in Chemistry and Science and Techniques of waste, now I teach Chemistry classes and also a class dealing with radioactive waste management in the Energy Engineering and Environment department. <br/> My research work at the Laboratory of Civil engineering and Environmental engineering (LGCIE) is currently aimed at the study of biophysicochemical interactions of pollutants in various compounds (soils, sediments, municipal solid waste) using molecular biology tools. I am very excited to take part in the iGEM 2012 project which leads to the development of a synergy, already launched, between the Biosciences department and Environmental sciences.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Yoann Louis<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Young ;) ) Associate professor at the Institut National des Sciences AppliquÈes de Lyon</p><br />
<p>Up to now my work is focused on the trace metal behavior in the environment and their interaction with dissolved organic matter in aquatic ecosystems.</p><br />
<p>Now my work at the LGCIE laboratory is mainly <strike>to develop a magic potion to be stronger than Asterix </strike>to study trace metals/ organic matter behavior in various waste and to give an expertise on their potential toxicity depending on the bio-physico-chemical conditions of the studied site.<br />
As a chemist, my interest in the iGEM project is to have a working approach angle enabling improvements of our environment thanks to our complementarity.</p><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/TeamTeam:Lyon-INSA/Team2012-10-26T01:25:16Z<p>Suxiaohui: </p>
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<h1>Our Team</h1><br />
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<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br />
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<h2>Students</h2><br />
<div class="wrapper"><br />
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<div id="studentListing"><br />
<div class="studentName"><br />
Alex Mizgier<br />
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<div class="studentName"><br />
Alexandre Duprey<br />
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<div class="studentName"><br />
Anne Haziza<br />
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<div class="studentName"><br />
Audrey Masi<br />
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<div class="studentName"><br />
Bastien Doix<br />
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Béryl Royer-Bertrand<br />
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<div class="studentName"><br />
Carine Gimbert<br />
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<div class="studentName"><br />
Clémence Gonthier<br />
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<div class="studentName"><br />
Ioana Sandu<br />
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Marion Traouan<br />
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<div class="studentName"><br />
Marion Wolfovski<br />
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Patricia Gifu<br />
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<div class="studentName"><br />
Rémi Hocq<br />
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<div class="studentName"><br />
Viviane Chansavang<br />
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Xavier Tholot<br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alex Mizgier<br />
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<img src="https://static.igem.org/mediawiki/2012/2/2f/Tetealexser.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/6/6c/AlexM_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Hairix, I am a third-year biochemist at INSA Lyon. I am from Chile, specifically from the northern limit of Western Patagonia.<br/><br />
I got into the iGEM project because I think that itís an unique experience and an excellent opportunity to learn more about bacterial manipulation and synthetic biology.<br/><br />
Apart from microbiology, I like reading, playing rugby, bicycle touring, and everything that involves nature and outdoor activities. You will find in me a knight-errant.<br/></p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alexandre Duprey<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/b/ba/AlexD.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/6/64/Alexmizgier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>This year, I'm back for another iGEM competition. I'm still at INSA Lyon, fourth-year biochemistry and biotechnology, and still interested in how we can regulate parts to make them work together. However this time I'm rather advising on the project with the intent to improve our weak points (who said modelling ?), and making sure the newcomers donít make (too many) mistakes. </br> Otherwise I play video games. A lot. Too much...</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Anne Haziza<br />
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<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p> I am a third-year student in Biochemistry and Biotechnologies at INSA Lyon. I am taking part in the iGEM competition for the first time. This project is a good way for me to improve my organization skills, ingenuity and dynamism which will be very useful for the future. </br>Outside of school, I enjoy cooking, playing handball and mainly traveling whenever possible! So I believe on the talent of her team to discover Amsterdam and of course Boston! </p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Audrey Masi<br />
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<img src="https://static.igem.org/mediawiki/2012/4/4f/Audre.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/e/e4/Audrey_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Squirrelix because I used to climb up on trees and hide in narrow cupboards to make jokes to my friends.<br><br />
I am a third-year bioengineer and I am joining the iGEM team after a first experience in molecular biology to improve my knowledge and discover the synthetic biology approach.<br><br />
Besides iGEM, I am interested in rock dancing, cooking, traveling, and above all that literature.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Bastien Doix<br />
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<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year student in Biochemistry and Biotechnology at INSA Lyon and taking part in iGEM for the first time. I am joining the iGEM team to have a first sight of what genetics and bacterial manipulation is and to step into something different from studies.</br> I like sports, skiing and computer graphics and hope to make it to Boston.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Béryl Royer-Bertrand<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>This is my second participation in the iGEM competition with the INSA Lyon team. In fourth year in Bioinformatics and Modeling, Iím helping this year the team in the modelling part from Scotland, where I am in academic exchange. </p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Carine Gimbert<br />
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<img src="https://static.igem.org/mediawiki/2012/d/dc/Carine.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/4/45/Carine_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Stoatix, it is my first participation in the INSA-Lyon team. In my previous studies, I worked on two projects about bioremediation and production of vanillin using biotechnology. So being part of the iGEM team is really important to continue learning how to manage bacteria and working in a student team. </br>I am fond of dance, photography and art in general. I live in a town where the international short film festival takes place : Clermont-Ferrand and one of my favorite is <a href="http://www.youtube.com/watch?v=DdLehwjV4pc">La révolution des crabesî</a></p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Clémence Gonthier<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>When you participate in iGEM once, you just want to take part in this great adventure again !<br><br />
In my fourth year of Biochemistry and Biotechnology, I'm still enjoying microbiology, modifying bacteria and creating new plasmids!<br><br />
I am this time on the advisor side... Helping the new team members searching for sponsors and guiding them for experiments in the lab.<br><br />
In my free time, I like dancing, playing the piano and traveling !</p><br />
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<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Ioana Sandu<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year Biochemistry student. I decided to join the Lyon team because I believe that the iGEM competition is a challenging experience that will put myself to the test. </br>Apart from synthetic biology, I like dancing, star-gazing and reading while listening to music (the older, the better).</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Traouan<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>I am an undergraduate student in biochemistry and biotechnologies at INSA Lyon. This is my first participation in an iGEM team. I consider this as an opportunity not only to acquire specific qualifications very useful for the future, but also to participate in the construction of an attractive project from scratch.<br/><br />
Besides my interest in biosciences, I am into sports like basket-ball or volley-ball, reading and cinema. I hope my "blondness" won't be an obstacle to the realization of this project.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Wolfovski<br />
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<img src="https://static.igem.org/mediawiki/2012/5/58/MarionW.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/8/8b/MarionW_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Rasberrix Olympix, I have a bachelor's degree in Sciences and I am now studying Biochemistry and Biotechnologies. This is my first participation in the iGEM competition. I love new challenges and iGEM is an amazing opportunity to manage together my passion for biology and building a project : a scientific adventure.</br> <br />
I love sports and making cookies for my lovely team !</p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Patricia Gifu<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am an undergraduate student in Biochemistry and Biotechnology at INSA de Lyon. This year, I participate for the first time in iGEM. I am very confident that INSA Lyon team is going to win because the team members worked very hard on the project and they all are skillful. I am passionate by biosciences. </br>Outside the school I like watching movies and traveling whenever I have time.</p><br />
</div> <br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Rémi Hocq<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8c/Remy.jpg/774px-Remy.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/thumb/1/10/Remy2.JPG/397px-Remy2.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am also known as Clumsix for often being Ö clumsy. Hell, I am the kind of guy who managed to cut my finger trying to open a bottle of beer and for the record, I was actually using a bottle-opener. My clumsiness is even contagious : GaÎl and Yoann also cut their finger during the summer.</br><br />
Anyway, I am also a third-year bioengineering student who love music (especially hard rock !), good food (though I am skinny and absolutely don't know why) and traveling. I may be a bit of a nerd too as I am a videogames fanatic.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Viviane Chansavang<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>iGEM rocks so badly, when you do it once, you just wanna do it again :) <br><br> <br />
But this year, I'm keeping a low profile, just helping out the newbies to settle in and feel comfy in the lab. I'm still enjoying working around bacteria, apart from <i>B. subtilis</i> producing lysostaphin, because it smells so badly you just wanna die when you happen to be working in the same room!<br><br><br />
I also like playing volleyball, cooking, baking, eating and above all traveling and meeting new people ^^ See y'all at the Jamboree! </p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Xavier Tholot<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year student in computer sciences at INSA Lyon. <br/><br />
I come from Clermont-Ferrand, the city where Michelin was created, which isn't far from Lyon.<br/><br />
I am fond of music (I play the guitar and the clarinet), and also of computers : this year, I joined the Lyon-INSA team to help them improving their wiki, and make it as great as possible.</p> <br />
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<br />
<h2>Advisors</h2><br />
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<div id="advisorListing"><br />
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Fanny Springer<br />
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<div class="advisorName"><br />
Gaël Chambonnier<br />
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<div class="advisorName"><br />
Philippe Thomas<br />
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<div class="advisorName"><br />
Romain Briandet<br />
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<div class="presentation adPres"><br />
<div class="nomPres"><br />
Fanny Springer<br />
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<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I've joined the Lyon-INSA team this year to see how we can change the world using genetically engineered material !</p><br />
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<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Gaël Chambonnier<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Hi everybody, here is Chambix!! I just graduated from the INSA Lyon in Biotechnologies but after two participations at the iGEM Competition, I couldnít do anything but continue my studies with a master in bioinformatics.<br><br />
After two years working in the lab for the team, I came back this year trying to help out the new team, sharing my experiences. Actually, they didnít really need my help. Theyíve done a great job with Bacillix. And for sure you'll have to deal with them in Amsterdam and Boston.</p><br />
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<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Philippe Thomas<br />
</div><br />
<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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<div class="textePres"><br />
<p>This year, Asterix and his friends have travelled until Buenos Aires in Argentina... They meet some strange people living riding horses, drinking mate, eating very good meat and raising cows in the infinite pampa! I'm one of this gauchos! I'm currently working in Buenos Aires for 18 months as an Engineer in Biotechnologies at Sanofi Pasteur. As it is not so easy to assist meeting and perform some experiments, the best for me was to be an advisor!</br><br />
I try to help out my Gallic friends when some extra work is needed and give my best as advisor, sharing my experiences of the iGEM competition with them! El hombre de la pampa can't wait to see the Lyon Biosciences Team rockiní Amsterdam and Boston with our very powerful bacteria! </p><br />
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</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Romain Briandet<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>I am the leader of the biofilm group at the INRA Micalis Institute. I have focused my research on microbial biofilms present in the food chain with special emphasis on their role in the persistence of pathogens. One of my main scientific line of interest is to identify the link between the 3D spatial organization of biofilms and the survival mechanisms of cells in face to the exposition of antimicrobials. Recently, my team reported that a tiny proportion of certain <i>Bacillus</i> species can tunnel through biofilms, creating pores that allow molecules to flow in. We are now evaluating the industrial benefit to pretreat target biofilms with swimming <i>Bacilli</i> cocktails to increase their efficacy and reduced their ecological impact.</p><br />
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<br />
<h2>Instructors</h2><br />
<div class="wrapper"><br />
<div id="instructorListing"><br />
<div class="instructorName"><br />
Agnès Rodrigue<br />
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<div class="instructorName"><br />
Corinne Dorel<br />
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<div class="instructorName"><br />
Olivier Brette<br />
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<div class="instructorName"><br />
Philippe Oger<br />
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<div class="instructorName"><br />
Valérie Desjardin<br />
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Yoann Louis<br />
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Agnès Rodrigue<br />
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<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>As an associate professor I find that iGEM is an unique experience, a place to mix teaching and practice in a long term project relying on studentsí motivation. My teaching interests are microbiology, molecular biology, protein engineering and bioinformatics. <br/> My research topic focus on understanding the molecular mechanisms involved in bacterial adaptation to a metal-rich environment, from molecular interactions to population biology. Apart from the basic approaches, this subject also offers opportunities to develop biological tools for bioremediation of spoiled environment or <i>in situ</i> detection of toxic compounds for instance, developments which I'm interested in. My motivation for iGEM is the same : conception and application. </p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Corinne Dorel<br />
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<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>My teaching activities in the Microbial Genetics mainly take place within the Biosciences department at INSA Lyon, but also at the Ecole Normale Supérieur de Lyon and at the University Claude Bernard Lyon 1.<br/><br/><br />
My research work is based on the understanding of genetic mechanisms involved in the formation of biofilms and the contamination of materials. Being the Communications officer for the Biosciences department, my participation in iGEM 2012 is part of a strategy to promote and share knowledge in the field of research in genetic engineering and more generally in Biological Sciences.</p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Olivier Brette<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
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<div class="textePres"><br />
<p>Associate Professor in economics at the Institut National des Sciences Appliquées (INSA) de Lyon</p><br />
<p>I teach the "economics of firm", "innovation economics", as well as the "economics of globalization" to engineering students.</p><br />
<p>I am affiliated with the CNRS Research Unit "Environment, City, Society" (EVS), where I pursue my research activities in theoretical and applied economics. From a theoretical viewpoint, my research work aims at developing the methodological, conceptual, as well as behavioral foundations of Institutional and Evolutionary Economics. I resort to this theoretical framework to deal with different kinds of issues in innovation economics and economic geography.</p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Philippe Oger<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Piezophilix because I work on high-pressure adaptation in microorganisms.<br />
My research is focussed on adaptation mechanisms in microbes from the deep-biosphere, and mainly in Archaea isolated from deep-sea hydrothermal vent systems.<br><br><br />
Our aim is to identify and quantify what genetic modifications make our favorite model, <i>Pyrococcus yanaosii</i>, require 500 times the atmospheric pressure for growth, when everybody else's favorite lab rat, <i>E. coli</i>, cannot even growth at the same hydrostatic pressure.<br><br><br />
My teaching activities at the University of Lyon deal with petroleum reservoir microbiology and the use of biosignatures for the study of past and present environments.</p><br />
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<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Valérie Desjardin<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Associate professor at the Institut National des Sciences Appliquées de Lyon</p><br />
<p>After a Master of Advanced Studies in Biochemistry and a PhD in Chemistry and Science and Techniques of waste, now I teach Chemistry classes and also a class dealing with radioactive waste management in the Energy Engineering and Environment department. <br/> My research work at the Laboratory of Civil engineering and Environmental engineering (LGCIE) is currently aimed at the study of biophysicochemical interactions of pollutants in various compounds (soils, sediments, municipal solid waste) using molecular biology tools. I am very excited to take part in the iGEM 2012 project which leads to the development of a synergy, already launched, between the Biosciences department and Environmental sciences.</p><br />
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<div class="presentation insPres"><br />
<div class="nomPres"><br />
Yoann Louis<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Young ;) ) Associate professor at the Institut National des Sciences AppliquÈes de Lyon</p><br />
<p>Up to now my work is focused on the trace metal behavior in the environment and their interaction with dissolved organic matter in aquatic ecosystems.</p><br />
<p>Now my work at the LGCIE laboratory is mainly <strike>to develop a magic potion to be stronger than Asterix </strike>to study trace metals/ organic matter behavior in various waste and to give an expertise on their potential toxicity depending on the bio-physico-chemical conditions of the studied site.<br />
As a chemist, my interest in the iGEM project is to have a working approach angle enabling improvements of our environment thanks to our complementarity.</p><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/TeamTeam:Lyon-INSA/Team2012-10-26T01:10:56Z<p>Suxiaohui: </p>
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<h1><br />
Our Team<br />
</h1><br />
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<div><center><b><big>Click on the title to show/hide the text.</big></b></center></div><br />
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<h2>Students</h2><br />
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<div class="studentName"><br />
Alex Mizgier<br />
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<div class="studentName"><br />
Alexandre Duprey<br />
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Anne Haziza<br />
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Audrey Masi<br />
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Bastien Doix<br />
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Béryl Royer-Bertrand<br />
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Carine Gimbert<br />
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<div class="studentName"><br />
Clémence Gonthier<br />
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Ioana Sandu<br />
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Marion Traouan<br />
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<div class="studentName"><br />
Marion Wolfovski<br />
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Patricia Gifu<br />
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<div class="studentName"><br />
Rémi Hocq<br />
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<div class="studentName"><br />
Viviane Chansavang<br />
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<div class="studentName"><br />
Xavier Tholot<br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alex Mizgier<br />
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<img src="https://static.igem.org/mediawiki/2012/2/2f/Tetealexser.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/6/6c/AlexM_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Hairix, I am a third-year biochemist at INSA Lyon. I am from Chile, specifically from the northern limit of Western Patagonia.<br/><br />
I got into the iGEM project because I think that itís an unique experience and an excellent opportunity to learn more about bacterial manipulation and synthetic biology.<br/><br />
Apart from microbiology, I like reading, playing rugby, bicycle touring, and everything that involves nature and outdoor activities. You will find in me a knight-errant.<br/></p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Alexandre Duprey<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/b/ba/AlexD.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/6/64/Alexmizgier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>This year, I'm back for another iGEM competition. I'm still at INSA Lyon, fourth-year biochemistry and biotechnology, and still interested in how we can regulate parts to make them work together. However this time I'm rather advising on the project with the intent to improve our weak points (who said modelling ?), and making sure the newcomers donít make (too many) mistakes. </br> Otherwise I play video games. A lot. Too much...</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Anne Haziza<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/9/94/P1030538.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p> I am a third-year student in Biochemistry and Biotechnologies at INSA Lyon. I am taking part in the iGEM competition for the first time. This project is a good way for me to improve my organization skills, ingenuity and dynamism which will be very useful for the future. </br>Outside of school, I enjoy cooking, playing handball and mainly traveling whenever possible! So I believe on the talent of her team to discover Amsterdam and of course Boston! </p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Audrey Masi<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4f/Audre.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/e/e4/Audrey_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Squirrelix because I used to climb up on trees and hide in narrow cupboards to make jokes to my friends.<br><br />
I am a third-year bioengineer and I am joining the iGEM team after a first experience in molecular biology to improve my knowledge and discover the synthetic biology approach.<br><br />
Besides iGEM, I am interested in rock dancing, cooking, traveling, and above all that literature.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Bastien Doix<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year student in Biochemistry and Biotechnology at INSA Lyon and taking part in iGEM for the first time. I am joining the iGEM team to have a first sight of what genetics and bacterial manipulation is and to step into something different from studies.</br> I like sports, skiing and computer graphics and hope to make it to Boston.</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Béryl Royer-Bertrand<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>This is my second participation in the iGEM competition with the INSA Lyon team. In fourth year in Bioinformatics and Modeling, Iím helping this year the team in the modelling part from Scotland, where I am in academic exchange. </p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Carine Gimbert<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/d/dc/Carine.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/4/45/Carine_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Stoatix, it is my first participation in the INSA-Lyon team. In my previous studies, I worked on two projects about bioremediation and production of vanillin using biotechnology. So being part of the iGEM team is really important to continue learning how to manage bacteria and working in a student team. </br>I am fond of dance, photography and art in general. I live in a town where the international short film festival takes place : Clermont-Ferrand and one of my favorite is <a href="http://www.youtube.com/watch?v=DdLehwjV4pc">La révolution des crabesî</a></p><br />
</div><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Clémence Gonthier<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>When you participate in iGEM once, you just want to take part in this great adventure again !<br><br />
In my fourth year of Biochemistry and Biotechnology, I'm still enjoying microbiology, modifying bacteria and creating new plasmids!<br><br />
I am this time on the advisor side... Helping the new team members searching for sponsors and guiding them for experiments in the lab.<br><br />
In my free time, I like dancing, playing the piano and traveling !</p><br />
</div><br />
</div><br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Ioana Sandu<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/a/a7/Ioana.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am a third-year Biochemistry student. I decided to join the Lyon team because I believe that the iGEM competition is a challenging experience that will put myself to the test. </br>Apart from synthetic biology, I like dancing, star-gazing and reading while listening to music (the older, the better).</p><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Traouan<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/2/2c/MarionT.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>I am an undergraduate student in biochemistry and biotechnologies at INSA Lyon. This is my first participation in an iGEM team. I consider this as an opportunity not only to acquire specific qualifications very useful for the future, but also to participate in the construction of an attractive project from scratch.<br/><br />
Besides my interest in biosciences, I am into sports like basket-ball or volley-ball, reading and cinema. I hope my "blondness" won't be an obstacle to the realization of this project.</p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Marion Wolfovski<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/58/MarionW.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8b/MarionW_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Rasberrix Olympix, I have a bachelor's degree in Sciences and I am now studying Biochemistry and Biotechnologies. This is my first participation in the iGEM competition. I love new challenges and iGEM is an amazing opportunity to manage together my passion for biology and building a project : a scientific adventure.</br> <br />
I love sports and making cookies for my lovely team !</p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Patricia Gifu<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am an undergraduate student in Biochemistry and Biotechnology at INSA de Lyon. This year, I participate for the first time in iGEM. I am very confident that INSA Lyon team is going to win because the team members worked very hard on the project and they all are skillful. I am passionate by biosciences. </br>Outside the school I like watching movies and traveling whenever I have time.</p><br />
</div> <br />
<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Rémi Hocq<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8c/Remy.jpg/774px-Remy.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/thumb/1/10/Remy2.JPG/397px-Remy2.JPG" width="180px"/><br />
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<div class="textePres"><br />
<p>I am also known as Clumsix for often being Ö clumsy. Hell, I am the kind of guy who managed to cut my finger trying to open a bottle of beer and for the record, I was actually using a bottle-opener. My clumsiness is even contagious : GaÎl and Yoann also cut their finger during the summer.</br><br />
Anyway, I am also a third-year bioengineering student who love music (especially hard rock !), good food (though I am skinny and absolutely don't know why) and traveling. I may be a bit of a nerd too as I am a videogames fanatic.</p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Viviane Chansavang<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>iGEM rocks so badly, when you do it once, you just wanna do it again :) <br><br> <br />
But this year, I'm keeping a low profile, just helping out the newbies to settle in and feel comfy in the lab. I'm still enjoying working around bacteria, apart from <i>B. subtilis</i> producing lysostaphin, because it smells so badly you just wanna die when you happen to be working in the same room!<br><br><br />
I also like playing volleyball, cooking, baking, eating and above all traveling and meeting new people ^^ See y'all at the Jamboree! </p><br />
</div><br />
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<br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Xavier Tholot<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>I am a third-year student in computer sciences at INSA Lyon. <br/><br />
I come from Clermont-Ferrand, the city where Michelin was created, which isn't far from Lyon.<br/><br />
I am fond of music (I play the guitar and the clarinet), and also of computers : this year, I joined the Lyon-INSA team to help them improving their wiki, and make it as great as possible.</p> <br />
</div><br />
</div><br />
</div><br />
<br />
<h2>Advisors</h2><br />
<div class="wrapper"><br />
<div id="advisorListing"><br />
<div class="advisorName"><br />
Fanny Springer<br />
</div><br />
<div class="advisorName"><br />
Gaël Chambonnier<br />
</div><br />
<div class="advisorName"><br />
Philippe Thomas<br />
</div><br />
<div class="advisorName"><br />
Romain Briandet<br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Fanny Springer<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I've joined the Lyon-INSA team this year to see how we can change the world using genetically engineered material !</p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Gaël Chambonnier<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
</div> <br />
</div><br />
<div class="textePres"><br />
<p>Hi everybody, here is Chambix!! I just graduated from the INSA Lyon in Biotechnologies but after two participations at the iGEM Competition, I couldnít do anything but continue my studies with a master in bioinformatics.<br><br />
After two years working in the lab for the team, I came back this year trying to help out the new team, sharing my experiences. Actually, they didnít really need my help. Theyíve done a great job with Bacillix. And for sure youíll have to deal with them in Amsterdam and Boston.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Philippe Thomas<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>This year, Asterix and his friends have travelled until Buenos Aires in Argentina... They meet some strange people living riding horses, drinking mate, eating very good meat and raising cows in the infinite pampa! Iím one of this gauchos! Iëm currently working in Buenos Aires for 18 months as an Engineer in biotechnology in Sanofi Pasteur. As it is not so easy to assist meeting and perform some experiments, the best for me was to be an advisor!</br><br />
I try to help out my Gallic friends when some extra work is needed and give my best as advisor, sharing my experiences of the iGEM competition with them! El hombre de la pampa canít wait to see the Lyon Biosciences Team rockiní Amsterdam and Boston with our very powerfull bacteria! </p><br />
</div><br />
</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Romain Briandet<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am the leader of the biofilm group at the INRA Micalis Institute. I have focused my research on microbial biofilms present in the food chain with special emphasis on their role in the persistence of pathogens. One of my main scientific line of interest is to identify the link between the 3D spatial organization of biofilms and the survival mechanisms of cells in face to the exposition of antimicrobials. Recently, my team reported that a tiny proportion of certain <i>Bacillus</i> species can tunnel through biofilms, creating pores that allow molecules to flow in. We are now evaluating the industrial benefit to pretreat target biofilms with swimming <i>Bacilli</i> cocktails to increase their efficacy and reduced their ecological impact.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<h2>Instructors</h2><br />
<div class="wrapper"><br />
<br />
<div id="instructorListing"><br />
<div class="instructorName"><br />
Agnès Rodrigue<br />
</div><br />
<div class="instructorName"><br />
Corinne Dorel<br />
</div><br />
<div class="instructorName"><br />
Olivier Brette<br />
</div><br />
<div class="instructorName"><br />
Philippe Oger<br />
</div><br />
<div class="instructorName"><br />
Valérie Desjardin<br />
</div><br />
<div class="instructorName"><br />
Yoann Louis<br />
</div><br />
<br />
<br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Agnès Rodrigue<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>As an associate professor I find that iGEM is an unique experience, a place to mix teaching and practice in a long term project relying on studentsí motivation. My teaching interests are microbiology, molecular biology, protein engineering and bioinformatics. <br/> My research topic focus on understanding the molecular mechanisms involved in bacterial adaptation to a metal-rich environment, from molecular interactions to population biology. Apart from the basic approaches, this subject offers also the opportunity to develop biological tools for the bioremediation of spoiled environment or the in situ detection of toxic compounds for instance, developments which Iím interested in. My motivation for iGEM is the same: conception and application. </p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Corinne Dorel<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>My teaching activities in the Microbial Genetics mainly take place within the Biosciences department at INSA Lyon, but also at the Ecole Normale SupÈrieur de Lyon and at the University Claude Bernard Lyon 1.<br/><br/><br />
My research work is based on the understanding of genetic mechanisms involved in the formation of biofilms and the contamination of materials. Being the Communications officer for the Biosciences department, my participation in iGEM 2012 is part of a strategy to promote and share knowledge in the field of research in genetic engineering and more generally in Biological Sciences.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Olivier Brette<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Associate Professor in economics at the Institut National des Sciences AppliquÈes (INSA) de Lyon</p><br />
<br />
<p>I teach the "economics of firm", "innovation economics", as well as the "economics of globalization" to engineering students.</p><br />
<br />
<p>I am affiliated with the CNRS Research Unit "Environment, City, Society" (EVS), where I pursue my research activities in theoretical and applied economics. From a theoretical viewpoint, my research work aims at developing the methodological, conceptual, as well as behavioral foundations of Institutional and Evolutionary Economics. I resort to this theoretical framework to deal with different kinds of issues in innovation economics and economic geography.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Philippe Oger<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
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<div class="textePres"><br />
<p>Also known as Piezophilix because I work on high-pressure adaptation in microorganisms.<br />
My research is focussed on adaptation mechanisms in microbes from the deep-biosphere, and mainly in Archaea isolated from deep-sea hydrothermal vent systems.<br><br><br />
Our aim is to identify and quantify what genetic modifications make our favorite model, <i>Pyrococcus yanaosii</i>, require 500 times the atmospheric pressure for growth, when everybody else's favorite lab rat, <i>E. coli</i>, cannot even growth at the same hydrostatic pressure.<br><br><br />
My teaching activities at the University of Lyon deal with petroleum reservoir microbiology and the use of biosignatures for the study of past and present environments.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Valérie Desjardin<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>Associate professor at the Institut National des Sciences AppliquÈes de Lyon</p><br />
<br />
<p>After a Master of Advanced Studies in Biochemistry and a PhD in Chemistry and Science and Techniques of waste, now I teach Chemistry classes and also a class dealing with radioactive waste management in the Energy Engineering and Environment department. <br/> My research work at the Laboratory of Civil engineering and Environmental engineering (LGCIE) is currently aimed at the study of biophysicochemical interactions of pollutants in various compounds (soils, sediments, municipal solid waste) using molecular biology tools. I am very excited to take part in the iGEM 2012 project which leads to the development of a synergy, already launched, between the Biosciences department and Environmental sciences.</p><br />
</div><br />
</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Yoann Louis<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>Young ;) ) Associate professor at the Institut National des Sciences AppliquÈes de Lyon</p><br />
<br />
<p>Up to now my work is focused on the trace metal behavior in the environment and their interaction with dissolved organic matter in aquatic ecosystems.</p><br />
<br />
<p>Now my work at the LGCIE laboratory is mainly <strike>to develop a magic potion to be stronger than Asterix </strike>to study trace metals/ organic matter behavior in various waste and to give an expertise on their potential toxicity depending on the bio-physico-chemical conditions of the studied site.<br />
As a chemist, my interest in the iGEM project is to have a working approach angle allowing the improvements of our environment thanks to our complementarity.</p><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/HPTeam:Lyon-INSA/HP2012-10-25T22:37:17Z<p>Suxiaohui: </p>
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<div class="introduction contenuTexte" style="margin:20px;font-size:15px;margin-top:0px;"><br />
<p>Our human practice project includes two themes addressing many very different questions.<br />
The first ones are about various intellectual property issues. The other theme relates to the popularization of sciences and more specifically to our contribution to the researchers’ night.</p><br />
<p>We have chosen to divide the theme "intellectual property" into two parts because this theme is the main part of our reflexion and it required a longer development, but also because we collaborated with the <a href="https://2012.igem.org/Team:British_Columbia">University of British Columbia</a> on this theme and an entire part seemed necessary to explain our work.<br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and is made up by a reflexion on the economic challenges the synthetic biology industry faces. The second one sums up our collaborative work on the intellectual property with the UBC iGEM team. And the third one quickly presents our scientific popularization involvement. </p><br><br />
<br><br />
<br />
<h2>Part I: Intellectual property issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br />
Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements:iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
<br><br />
<br><br />
Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
<br><br />
<br />
The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Common's system", which will be discussed further on. We will assume the iGEM team members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:70%;"><br />
We will most notably refer to both Joseph Stiglitz’s (Nobel prize in economics in 2001) and Elinor Ostrom’s (Nobel prize in economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
<br><br />
<br/><br />
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<h3>The patent system: an efficient economic model ?</h3><br />
<br />
<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first one to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
<br><br />
Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted invention have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
<br><br />
A patent is granted:<br><br />
<ul><br />
<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
</ul><br />
<br><br />
<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
<br><br />
The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even the genetically modified bacterias may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 80s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy.).<br><br />
<br> <br />
The goal of this brief study though, is not to discuss this matter.<br><br />
<br><br />
<div class="petitSsTitre">Why patents have been created?</div><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new age for intellectual property began.<br><br />
<br><br />
<br><br />
Instead, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
</div><br />
<br><br />
<br><br />
Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patent’s creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
<br />
<br />
<br/><br />
As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
<br><br />
<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
<br><br />
<br><br />
For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components.When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
<br><br />
Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:65%;"><br />
As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licences of many expensive patents from the incumbent firms.<br><br />
<br><br />
<br><br />
Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief resume. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ?. Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such, e.g., as shown in inventions and discoveries of more and better ways and means, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
</div><br />
<br><br />
This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
<br><br />
Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing.In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
<br><br />
<div style="margin:30px;"><br />
<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
</div><br />
<br><br />
<br><br />
<br />
In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation.The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to embark on a collective reflexion on its contribution to the development of a "synthetic biology commonns”.<br />
<br />
<br><br />
<br><br />
<h3>The Commons: an alternative model</h3><br />
<br><br />
<br><br />
<br />
The open source software's users have been in ever greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies 92% used open source software during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
<br><br />
<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license.This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
<br />
<br><br />
<br><br />
The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
<br><br />
<br><br />
However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel prize in economics in 2009, broadened the economists' mind on this particular topic.<br />
<br><br />
<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th century.<br />
<br><br />
<br><br />
Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches: each community managing a commons fixes its own rules by defining a governance frameworkl and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
<br />
<br><br />
<br><br />
Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits.<br />
<br />
<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
<br />
<li>rules in use are well matched to local needs and conditions</li><br />
<br />
<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
<br />
<li>the right of community members to devise their own rules is respected by external authorities</li><br />
<br />
<li>a system for self-monitoring members’ behavior has been established</li> <br />
<br />
<li>a graduated system of sanctions is available</li> <br />
<br />
<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
<br><br />
Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
<br><br />
The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic value of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
<br><br />
<br><br />
<br />
<h3>Bibliography</h3><br />
<br><br />
<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
<br />
<br />
</div><br />
</div><br />
<br />
<h2>Part II: FAQ</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br><br />
Our adventure in Amsterdam made us realize how abstract our reflexion was. We decided then to deepen it in a more concrete way through this FAQ which was inspired by the very questions we were asked in Holland.<br />
<br><br />
<br><br />
<i>Is it entirely impossible to patent an invention based on a public Part ?</i><br />
<br><br />
<br><br />
First of all, we shall remind the patent granting requirements :<br />
<br><br />
<br><br />
<li>the novelty condition (i.e. the invention must be partially or totally new);</li><br />
<li>the non-obviousness condition (U.S. patent law) or the inventiveness condition (in European patent law);</li><br />
<li>the usefulness condition (U.S. patent law) or the industrial applicability condition (in European patent law).</li><br />
<br><br />
<br />
<br />
Obviously, patenting public parts is impossible considering the first condition. However novel devices using public parts may be considered for protection through patents, depending on the degree of novelty carried by this device<br />
<br><br />
<br><br />
<br />
<i>Could private actors like companies belong to and participate to a “synthetic biology commons” ?</i><br><br><br />
<br />
Fact : companies can take part in a commons. Many companies (Small and Medium Enterprises as well as big companies) currently do make profit in the open source software industry. This opportunity is not confined to the software industry: the idea of an “open source biology” has also emerged. This movement, also referred to as “open access biology”, has appeared in bio-industries such as the pharmaceutical sector, in most cases to prevent other companies to patent their research. The SNP Consortium, founded in 1999, gathers 10 pharmaceutical companies which decided to release some of their data concerning the human genome in the public domain. A few other more recent examples, including Pfizer (2006), Novartis (2007) and Syngenta (2002), tend to show that sharing knowledge has become part of the economic strategies in biology (Henkel & Maurer, 2007).<br><br />
<br><br />
Besides, such a sharing knowledge strategy allows companies to make good use of free resources, the validity of which has already been controlled by a (potentially) large community. Moreover, many business models may be designed (and have already been implemented to some extent), which are based on indirect profits, such as selling services for example.<br><br />
<br><br />
More generally, the ability of a commons to create value mainly derives from its capacity to link together different kinds of actors (companies, scholars, hackers, etc.) pursuing different objectives : making profits, publishing, or more original motivations, such as "the fun of programming", "the sense of belonging to a common culture, where participants share a common ideology, often characterized by reciprocity" (Henry and Stiglitz, 2010).<br />
<br><br />
<br><br />
<i>Can a commons coexist with other forms of the organization of economic activities (such as the market or the state involvement) inside the same sector ?</i><br />
<br><br />
<br><br />
Nothing opposes the possibility that different modes of coordination co-exist within the same sector. A given firm may share knowledge in a commons perspective, while simultaneously developing market relations. For instance, some software vendors sell proprietary software (“proprietary bricks”) that complement a main open source program (which one is free to use) like an option.<br><br />
<br><br />
Existing economic associations such as the BiOS (“Biological innovation for an Open Society”) Initiative of Cambia, suggest that such a combination would be possible in the field of synthetic biology. The BiOS license allows the licensees to use Cambia enabling technology (a few key plant gene transfer patented technologies) royalty-free, on condition that the improvements made to the technology are also made freely available (i.e. they can be used royalty-free by other licensees). Furthermore, the BiOS founders, who claim that they have adapted the “open source” approach to biology (the source code being here the enabling technology), also argue that their licensing device does not lessen the usual incentives to innovate, including the possibility of patenting some products developed from the application of the enabling technology. <br><br />
<br><br />
<br />
<br />
<i>Why try to find our own rules for the iGEM commons ? The open source seems to work great, so shouldn’t we use theirs ?</i><br />
<br><br />
<br><br />
Ostrom put forward a set of generic principles for a commons to be economically viable. One of them is that every single commons has its own rules that are defined by both its actors and the resources that are dealt with.<br />
Not only are the actors of the open source software movement different from the synthetic biology community but also the code source is a resource different from the biological parts.These reasons make it hardly possible that the same rules could be applied to both commons, especially on the legal point of view. Indeed, the open source software movement is based on the spreading of open source licenses, which derive from copyright (“copyleft”). Such a solution is generally considered by legal experts as unadapted to biological parts (Henkel & Maurer, 2007). These experts also suggest that an economic model combining patents with legal devices ensuring free knowledge sharing and use would be the best way to the economic development of synthetic biology and to the spreading of innovations.<br />
<br><br />
<br><br />
<br />
<i>Should not we consider that the bases of a synthetic biology commons have already been defined, by several organisms, including the iGEM Foundation itself ?</i><br />
<br><br />
<br><br />
<br />
The iGEM community has already some attributes of a commons. However, we consider that this commons could be better structured and could play a far bigger role in orienting the development of synthetic biology.<br />
The objectives of our initiative are threefold :<br />
<br><br />
<br><br />
<li>first, leading every member of the iGEM community to be aware of participating to a commons;</li><br><br />
<li>second, provoking a debate within the iGEM community to precise its founding rules. The community should collectively deliberate on the definition of a set of rules regarding the rights to access BioBricks, to use them, to control their application, to impose sanctions if necessary, etc.;</li><br><br />
<li>third, leading the iGEM community to discuss its place and the role it may play in the development of an overall synthetic biology commons, involving different kinds of actors : other associations such as the BiOS Initiative, public research units and higher education organizations, companies, and so on. Though building an overall synthetic biology commons is far from being an easy task, it seems to be a growing concern for many actors, including public authorities. For instance, the current Minister of Higher Education and Research in France, Geneviève Fioraso, recently wrote a comprehensive Parliamentary Report on the scientific, technological and economic stakes of synthetic biology, which was published a few months before becoming a Minister (Fioraso Report, 2012). This report notably highlights the crucial role of iGEM in the development of synthetic biology.</li><br><br />
<br />
<br><br />
<br><br />
<h3>Bibliography</h3><br />
<br><br />
We used all the previous references (q.v. part I), though we added : <br><br />
<ul><br />
<li>Rai A, Boyle J (2007) <em>Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons.</em> PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058</li><br><br />
<li>Bios website : <a href="http://www.bios.net/daisy/bios/home.html">Bios website</a></li><br><br />
<li> Henkel J, Maurer S (2007) <em>The economics of synthetic biology</em>. Mol Syst Biol 3: 117. </li><br><br />
<li> Fioraso G (2012) <em>Les enjeux de la biologie de synthèse</em>. Report for the "Office parlementaire d'évaluation des choix scientifiques et technologiques".<br />
</ul><br />
<br><br><br />
</div><br />
</div><br />
<br />
<h2>Part III: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey.281 iGEMers had answered the survey when we treated it .<br />
<br><br />
This collaboration allowed us to broaden our reflexion to iGEM teams' issues.<br />
<br><br />
<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly we studied past iGEMers experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
<br><br />
Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
<br><br />
Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may results in a patent experience if the materials used are under a proprietary license. <br />
<br><br />
Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers IP experiences.<br />
<br/><br />
<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what was its nature. The results are that most of them (46 %) had already used patented materials before. However, 15% renounced to actually use them, which shows how patent can impede research and innovation: getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
<br><br />
The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10 % of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
<br><br />
Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
<br><br />
Our sole conclusion on this matter is that the IPR often does affect iGEM projects, even if in general the patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
<br />
<br><br />
It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
<br><br />
Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
<br><br />
To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member or / and to a faculty advisor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
<br><br />
<br><br />
<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the intellectual property rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks .<br />
<br><br />
Besides, this survey deepened our main reflexion (q.v. part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the intellectual property rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
</div><br />
</div><br />
<br />
<h2>Part IV: Popularization of Science</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
Stereotypes and various pieces of information about science are spread in such a way that people sometimes find it hard to decipher the truth about everything that is said. The public, but also non-biologist scientists, may sometimes have to look for a reliable source to make their own opinion. <br><br />
We are convinced that we have a role to play and have decided to involve ourselves in several actions.<br />
<br><br />
<br><br />
<h3>First, we organized three conferences with cutting-edge experts:</h3><br />
<ul><br />
<li>“Bacterial swimmers can penetrate biofilms, making them vulnerable to destruction.” by Romain Briandet, expert in Surface Hygiene/Bioadhesion in May 2012.<br />
This meeting enabled the Lyon-INSA iGEM team to discover the amazing properties of Bacillus subtilis swimmers and their potential as a tool for biofilm control.</li><br />
<li>“Synthetic Biology : Biotechnologies revival?” by François Képès, national and international SynBio expert in September 2012. Képès initiated a debate on a paradox that biotechnologies raise. Paradoxically, biotechnologies aim at improving the industrialization of their products with normalization, but also intend to be more creative by setting free from existing constraints.</li><br />
<li>Olivier Brette in September 2012. The stakes of intellectual property, science and innovation were presented and discussed with the assembly of students and staff from various scientific fields (mechanics, informatics, economic intelligence, ethics…).</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Then, we organized meetings with non-biologist scientists and the public:</h3><br />
<ul><br />
<li>From May to September 2012 : Open debates on the stakes of SynBio with staff and students from Mechanics, Informatics and Telecoms INSA departments but also staff from INSAVALOR (Research and Development, Research Valuation of INSA-affiliated company) through the “Filière Ingénieur-Entreprendre” (Entrepreneurship formation). Participation to the iGEM Entrepreneurship Division was even considered.</li><br />
<li>September 2012 : The Researchers' Night. Since 7 years, this event has been taking place on a single September night in about 300 cities all over Europe. The main goal of this night is to put researchers in touch with the public. Thus, they can explain what they are doing, how and what can be the applications in the day-to-day life.</li><br />
</ul><br />
<br><br />
<br><br />
<h3>More about the Researchers' Night</h3><br />
This year, the theme was "Imagine the future": what future will emerge from research laboratories? How do scientists imagine the future? Archeology, Physics, Philosophy, Biosciences or Linguistic: researchers shared their lab experiences and thoughts on the future. Therefore, in order to defend a controversial discipline, Synthetic Biology (a.k.a. SynBio), the Lyon-INSA team members participated in the Researchers’ Night, which was held on September 28th, 2012, at the CCO of Villeurbanne.<br />
<br><br />
<br><br />
Indeed, an article published by a French team: “Séralini et al., Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize, Food and Chemical Toxicology, Volume 50, Issue 11, November 2012, Pages 4221–4231” and also in the paper “Le Monde” on September 25th, 2012, on deleterious effect of transgenic maize on rats, had a dramatic effect on the public opinion in Europe. Thus, to illustrate that SynBio can be used to improve the environment, health and life of people, we decided to explain the potential of SynBio and to exchange with the public through a scientific speed-dating and around a table containing diagrams and Petri dishes.<br />
<br><br />
<br><br />
“What is it?” asked a little boy. “What are you doing?” asked a woman. We could introduce them to our friends: bacteria! Then, one of the iGEM team members answered: “Bacteria are little organisms which grow, eat and die like every living being. These bacteria are not always pathogenic and scientists can improve their qualities with the introduction of DNA fragments containing genes.”<br />
<br><br />
<br><br />
So the objective is reached: we aroused the surprise, the questioning and the amazement of people. In this respect, we pointed out the usefulness of SynBio. We decided to focus on medical application of BABS (Bacteria Improved by SynBio) such as insulin, growth hormones, and artemisinin production, and also on the food industry application with food coloring or artificial flavor production that already work.<br />
<br><br />
<br><br />
Of course, we have also presented our project as an innovative solution to kill and disperse biofilm in all pipelines or tanks and to prevent any colonization of bacteria.<br />
<br><br />
<br><br />
<br />
</div><br />
</div><br />
<br />
<h2>Part V: Public sensitization</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
To have an idea of how the public would react to the use of our solution in cleaning the installation for food and cosmetic industries, we have sent a survey in our school. We have received about 930 answers in less than a week, proving the interest people is showing on our subject.<br />
<br><br />
<br><br />
<h3>What are the public thoughts on the use of bacteria in cleaning process?</h3><br />
<br><br />
<br><br />
Studied population : <br />
<br><br />
<br><br />
We choose to interview a specific population : The larger part is constituted by scientific students who are from 18 to 24 years old .<br />
<br><br />
<br> <br />
<center><img src="https://static.igem.org/mediawiki/2012/a/ab/Age1.png" width="70%"/></center><br />
<br><br />
<br><br />
Moreover, their studies, knowledges or interests in biology are different. The survey allows us to precise a tendency on the acceptance of bacteria in food and cosmetic industries by the young population : the future consumers. Furthermore, it gives us an idea whether our project would be workable and marketable or not.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/de/Biology1.png" width="70%"/></center><br />
<br><br />
<br><br />
Results :<br />
<br><br />
<br><br />
First, we interest ourselves in the attention the public pays to the composition of the products they use in every day life.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c9/Concerned.png" width="70%"/></center><br />
<br><br />
<br><br />
In general, ¾ of the population is interested and concerned about what is put in their products. So, it seems relevant to ask them what they would accept or not.<br />
Foods and cosmetics are the products, people are more likely in contact with. Besides, they are industries we aim to clean with our bacteria.<br />
The important is to know the opinion of the larger public on this matter :<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/f/f5/Agecomparison.png" width="70%"/></center><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c5/Comparrsoncosm.png" width="70%"/></center><br />
remark : the bacteria are indicated as eliminated after cleaning and never in direct contact with the products.<br />
<br><br />
<br><br />
As we could expect, the biological substances are the ones people are more willing to accept, both in food and cosmetic industries.<br />
Moreover, There are not real concerns for the use of living bacteria in these two fields.<br />
<br><br />
<br><br />
The questions of the chemicals and the GMO’s is quiet different : they are more accepted in cosmetics than in food (in general people seem more precocious on what they eat..) but generally less well see.<br />
The most remarkable is that the interviewed people have almost the same “reluctance” for the use of chemicals and GMOs. That can be interpreted as the students'sensitization of the negative effects of chemicals.<br />
<br><br />
<br><br />
We find interesting to gather the surveyed by age groups : <br />
<br><br />
Some of the surveyed people belong to the 0-17 years old age group, and few to the more than 35 years old. The analysis of their answers leads us to note that the group above 35 years old has a tendency to refuse GMOs and to accept more easily living bacteria and chemicals in their products. <br />
On the contrary, the youngest are more reluctant about chemicals in their foods, and least cautious on what is put in their cosmetics.<br />
<br><br />
<br><br />
Through this survey, we remark no real issues to the public acceptance of Biofilm Killer in food and cosmetic industries. In fact, thanks to these results, we even feel an inducement in our search to limit the use of chemicals and a public aprovement of our Biofilm Killer.<br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="introduction contenuTexte" style="margin:20px;font-size:15px;"><br />
<p><br />
<em> Special thanks to our faculty advisor O. Brette for his advice and for his working late to help us and to the UBC team for sharing its poll results with ours. </em><br />
</p><br />
<br><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/HPTeam:Lyon-INSA/HP2012-10-25T22:13:53Z<p>Suxiaohui: </p>
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<div class="introduction contenuTexte" style="margin:20px;font-size:15px;margin-top:0px;"><br />
<p>Our human practice project includes two themes addressing many very different questions.<br />
The first ones are about various intellectual property issues. The other theme relates to the popularization of sciences and more specifically to our contribution to the researchers’ night.</p><br />
<p>We have chosen to divide the theme "intellectual property" into two parts because this theme is the main part of our reflexion and it required a longer development, but also because we collaborated with the <a href="https://2012.igem.org/Team:British_Columbia">University of British Columbia</a> on this theme and an entire part seemed necessary to explain our work.<br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and is made up by a reflexion on the economic challenges the synthetic biology industry faces. The second one sums up our collaborative work on the intellectual property with the UBC iGEM team. And the third one quickly presents our scientific popularization involvement. </p><br><br />
<br><br />
<br />
<h2>Part I: Intellectual property issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br />
Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements:iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
<br><br />
<br><br />
Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
<br><br />
<br />
The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Common's system", which will be discussed further on. We will assume the iGEM team members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:70%;"><br />
We will most notably refer to both Joseph Stiglitz’s (Nobel prize in economics in 2001) and Elinor Ostrom’s (Nobel prize in economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
<br><br />
<br/><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/4/4e/Patent.gif" class="fancyable"><br />
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<h3>The patent system: an efficient economic model ?</h3><br />
<br />
<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first one to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
<br><br />
Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted invention have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
<br><br />
A patent is granted:<br><br />
<ul><br />
<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
</ul><br />
<br><br />
<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
<br><br />
The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even the genetically modified bacterias may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 80s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy.).<br><br />
<br> <br />
The goal of this brief study though, is not to discuss this matter.<br><br />
<br><br />
<div class="petitSsTitre">Why patents have been created?</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/8f/Droits_homme.jpg" class="fancyable"><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new age for intellectual property began.<br><br />
<br><br />
<br><br />
Instead, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
</div><br />
<br><br />
<br><br />
Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patent’s creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
<br />
<br />
<br/><br />
As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
<br><br />
<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
<br><br />
<br><br />
For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components.When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
<br><br />
Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:65%;"><br />
As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licences of many expensive patents from the incumbent firms.<br><br />
<br><br />
<br><br />
Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief resume. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ?. Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such, e.g., as shown in inventions and discoveries of more and better ways and means, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
</div><br />
<br><br />
This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
<br><br />
Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing.In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
<br><br />
<div style="margin:30px;"><br />
<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
</div><br />
<br><br />
<br><br />
<br />
In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation.The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to embark on a collective reflexion on its contribution to the development of a "synthetic biology commonns”.<br />
<br />
<br><br />
<br><br />
<h3>The Commons: an alternative model</h3><br />
<br><br />
<br><br />
<br />
The open source software's users have been in ever greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies 92% used open source software during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
<br><br />
<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license.This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
<br />
<br><br />
<br><br />
The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
<br><br />
<br><br />
However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel prize in economics in 2009, broadened the economists' mind on this particular topic.<br />
<br><br />
<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th century.<br />
<br><br />
<br><br />
Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches: each community managing a commons fixes its own rules by defining a governance frameworkl and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
<br />
<br><br />
<br><br />
Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits.<br />
<br />
<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
<br />
<li>rules in use are well matched to local needs and conditions</li><br />
<br />
<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
<br />
<li>the right of community members to devise their own rules is respected by external authorities</li><br />
<br />
<li>a system for self-monitoring members’ behavior has been established</li> <br />
<br />
<li>a graduated system of sanctions is available</li> <br />
<br />
<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
<br><br />
Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
<br><br />
The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic value of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
<br><br />
<br><br />
<br />
<h3>Bibliography</h3><br />
<br><br />
<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
<br />
<br />
</div><br />
</div><br />
<br />
<h2>Part II: FAQ</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br><br />
Our adventure in Amsterdam made us realize how abstract our reflexion was. We decided then to deepen it in a more concrete way through this FAQ which was inspired by the very questions we were asked in Holland.<br />
<br><br />
<br><br />
<i>Is it entirely impossible to patent an invention based on a public Part ?</i><br />
<br><br />
<br><br />
First of all, we shall remind the patent granting requirements :<br />
<br><br />
<br><br />
<li>the novelty condition (i.e. the invention must be partially or totally new);</li><br />
<li>the non-obviousness condition (U.S. patent law) or the inventiveness condition (in European patent law);</li><br />
<li>the usefulness condition (U.S. patent law) or the industrial applicability condition (in European patent law).</li><br />
<br><br />
<br />
<br />
Obviously, patenting public parts is impossible considering the first condition. However novel devices using public parts may be considered for protection through patents, depending on the degree of novelty carried by this device<br />
<br><br />
<br><br />
<br />
<i>Could private actors like companies belong to and participate to a “synthetic biology commons” ?</i><br><br><br />
<br />
Fact : companies can take part in a commons. Many companies (Small and Medium Enterprises as well as big companies) currently do make profit in the open source software industry. This opportunity is not confined to the software industry: the idea of an “open source biology” has also emerged. This movement, also referred to as “open access biology”, has appeared in bio-industries such as the pharmaceutical sector, in most cases to prevent other companies to patent their research. The SNP Consortium, founded in 1999, gathers 10 pharmaceutical companies which decided to release some of their data concerning the human genome in the public domain. A few other more recent examples, including Pfizer (2006), Novartis (2007) and Syngenta (2002), tend to show that sharing knowledge has become part of the economic strategies in biology (Henkel & Maurer, 2007).<br><br />
<br><br />
Besides, such a sharing knowledge strategy allows companies to make good use of free resources, the validity of which has already been controlled by a (potentially) large community. Moreover, many business models may be designed (and have already been implemented to some extent), which are based on indirect profits, such as selling services for example.<br><br />
<br><br />
More generally, the ability of a commons to create value mainly derives from its capacity to link together different kinds of actors (companies, scholars, hackers, etc.) pursuing different objectives : making profits, publishing, or more original motivations, such as "the fun of programming", "the sense of belonging to a common culture, where participants share a common ideology, often characterized by reciprocity" (Henry and Stiglitz, 2010).<br />
<br><br />
<br><br />
<i>Can a commons coexist with other forms of the organization of economic activities (such as the market or the state involvement) inside the same sector ?</i><br />
<br><br />
<br><br />
Nothing opposes the possibility that different modes of coordination co-exist within the same sector. A given firm may share knowledge in a commons perspective, while simultaneously developing market relations. For instance, some software vendors sell proprietary software (“proprietary bricks”) that complement a main open source program (which one is free to use) like an option.<br><br />
<br><br />
Existing economic associations such as the BiOS (“Biological innovation for an Open Society”) Initiative of Cambia, suggest that such a combination would be possible in the field of synthetic biology. The BiOS license allows the licensees to use Cambia enabling technology (a few key plant gene transfer patented technologies) royalty-free, on condition that the improvements made to the technology are also made freely available (i.e. they can be used royalty-free by other licensees). Furthermore, the BiOS founders, who claim that they have adapted the “open source” approach to biology (the source code being here the enabling technology), also argue that their licensing device does not lessen the usual incentives to innovate, including the possibility of patenting some products developed from the application of the enabling technology. <br><br />
<br><br />
<br />
<br />
<i>Why try to find our own rules for the iGEM commons ? The open source seems to work great, so shouldn’t we use theirs ?</i><br />
<br><br />
<br><br />
Ostrom put forward a set of generic principles for a commons to be economically viable. One of them is that every single commons has its own rules that are defined by both its actors and the resources that are dealt with.<br />
Not only are the actors of the open source software movement different from the synthetic biology community but also the code source is a resource different from the biological parts.These reasons make it hardly possible that the same rules could be applied to both commons, especially on the legal point of view. Indeed, the open source software movement is based on the spreading of open source licenses, which derive from copyright (“copyleft”). Such a solution is generally considered by legal experts as unadapted to biological parts (Henkel & Maurer, 2007). These experts also suggest that an economic model combining patents with legal devices ensuring free knowledge sharing and use would be the best way to the economic development of synthetic biology and to the spreading of innovations.<br />
<br><br />
<br><br />
<br />
<i>Should not we consider that the bases of a synthetic biology commons have already been defined, by several organisms, including the iGEM Foundation itself ?</i><br />
<br><br />
<br><br />
<br />
The iGEM community has already some attributes of a commons. However, we consider that this commons could be better structured and could play a far bigger role in orienting the development of synthetic biology.<br />
The objectives of our initiative are threefold :<br />
<br><br />
<br><br />
<li>first, leading every member of the iGEM community to be aware of participating to a commons;</li><br><br />
<li>second, provoking a debate within the iGEM community to precise its founding rules. The community should collectively deliberate on the definition of a set of rules regarding the rights to access BioBricks, to use them, to control their application, to impose sanctions if necessary, etc.;</li><br><br />
<li>third, leading the iGEM community to discuss its place and the role it may play in the development of an overall synthetic biology commons, involving different kinds of actors : other associations such as the BiOS Initiative, public research units and higher education organizations, companies, and so on. Though building an overall synthetic biology commons is far from being an easy task, it seems to be a growing concern for many actors, including public authorities. For instance, the current Minister of Higher Education and Research in France, Geneviève Fioraso, recently wrote a comprehensive Parliamentary Report on the scientific, technological and economic stakes of synthetic biology, which was published a few months before becoming a Minister (Fioraso Report, 2012). This report notably highlights the crucial role of iGEM in the development of synthetic biology.</li><br><br />
<br />
<br><br />
<br><br />
<h3>Bibliography</h3><br />
<br><br />
We used all the previous references (q.v. part I), though we added : <br><br />
<ul><br />
<li>Rai A, Boyle J (2007) <em>Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons.</em> PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058</li><br><br />
<li>Bios website : <a href="http://www.bios.net/daisy/bios/home.html">Bios website</a></li><br><br />
<li> Henkel J, Maurer S (2007) <em>The economics of synthetic biology</em>. Mol Syst Biol 3: 117. </li><br><br />
<li> Fioraso G (2012) <em>Les enjeux de la biologie de synthèse</em>. Report for the "Office parlementaire d'évaluation des choix scientifiques et technologiques".<br />
</ul><br />
<br><br><br />
</div><br />
</div><br />
<br />
<h2>Part III: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey.281 iGEMers had answered the survey when we treated it .<br />
<br><br />
This collaboration allowed us to broaden our reflexion to iGEM teams' issues.<br />
<br><br />
<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly we studied past iGEMers experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
<br><br />
Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
<br><br />
Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may results in a patent experience if the materials used are under a proprietary license. <br />
<br><br />
Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers IP experiences.<br />
<br/><br />
<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what was its nature. The results are that most of them (46 %) had already used patented materials before. However, 15% renounced to actually use them, which shows how patent can impede research and innovation: getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
<br><br />
The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10 % of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
<br><br />
Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
<br><br />
Our sole conclusion on this matter is that the IPR often does affect iGEM projects, even if in general the patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
<br />
<br><br />
It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
<br><br />
Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
<br><br />
To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member or / and to a faculty advisor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
<br><br />
<br><br />
<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the intellectual property rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks .<br />
<br><br />
Besides, this survey deepened our main reflexion (q.v. part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the intellectual property rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
</div><br />
</div><br />
<br />
<h2>Part IV: Popularization of Science</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
Stereotypes and various pieces of information about science are spread in such a way that people sometimes find it hard to decipher the truth about everything that is said. The public, but also non-biologist scientists, may sometimes have to look for a reliable source to make their own opinion. <br><br />
We are convinced that we have a role to play and have decided to involve ourselves in several actions.<br />
<br><br />
<br><br />
<h3>First, we organized three conferences with cutting-edge experts:</h3><br />
<ul><br />
<li>“Bacterial swimmers can penetrate biofilms, making them vulnerable to destruction.” by Romain Briandet, expert in Surface Hygiene/Bioadhesion in May 2012.<br />
This meeting enabled the Lyon-INSA iGEM team to discover the amazing properties of Bacillus subtilis swimmers and their potential as a tool for biofilm control.</li><br />
<li>“Synthetic Biology : Biotechnologies revival?” by François Képès, national and international SynBio expert in September 2012. Képès initiated a debate on a paradox that biotechnologies raise. Paradoxically, biotechnologies aim at improving the industrialization of their products with normalization, but also intend to be more creative by setting free from existing constraints.</li><br />
<li>Olivier Brette in September 2012. The stakes of intellectual property, science and innovation were presented and discussed with the assembly of students and staff from various scientific fields (mechanics, informatics, economic intelligence, ethics…).</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Then, we organized meetings with non-biologist scientists and the public.</h3><br />
<ul><br />
<li>From May to September 2012 : Open debates on the stakes of SynBio with staff and students from Mechanics, Informatics and Telecoms INSA departments but also staff from INSAVALOR (Research and Development, Research Valuation of INSA-affiliated company) through the “Filière Ingénieur-Entreprendre” (Entrepreneurship formation). Participation to the iGEM Entrepreneurship Division was even considered.</li><br />
<li>September 2012 : The Researchers' Night. Since 7 years, this event has been taking place on a single September night in about 300 cities all over Europe. The main goal of this night is to put researchers in touch with the public. Thus, they can explain what they are doing, how and what can be the applications in the day-to-day life.</li><br />
</ul><br />
<br><br />
<br><br />
<h3>More about the Researchers' Night</h3><br />
This year, the theme was "Imagine the future": what future will emerge from research laboratories? How do scientists imagine the future? Archeology, Physics, Philosophy, Biosciences or Linguistic: researchers shared their lab experiences and thoughts on the future. Therefore, in order to defend a controversial discipline, Synthetic Biology (a.k.a. SynBio), the Lyon-INSA team members participated in the Researchers’ Night, which was held on September 28th, 2012, at the CCO of Villeurbanne.<br />
<br><br />
<br><br />
Indeed, an article published by a French team: “Séralini et al., Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize, Food and Chemical Toxicology, Volume 50, Issue 11, November 2012, Pages 4221–4231” and also in the paper “Le Monde” on September 25th, 2012, on deleterious effect of transgenic maize on rats, had a dramatic effect on the public opinion in Europe. Thus, to illustrate that SynBio can be used to improve the environment, health and life of people, we decided to explain the potential of SynBio and to exchange with the public through a scientific speed-dating and around a table containing diagrams and Petri dishes.<br />
<br><br />
<br><br />
“What is it?” asked a little boy. “What are you doing?” asked a woman. We could introduce them to our friends: bacteria! Then, one of the iGEM team members answered: “Bacteria are little organisms which grow, eat and die like every living being. These bacteria are not always pathogenic and scientists can improve their qualities with the introduction of DNA fragments containing genes.”<br />
<br><br />
<br><br />
So the objective is reached: we aroused the surprise, the questioning and the amazement of people. In this respect, we pointed out the usefulness of SynBio. We decided to focus on medical application of BABS (Bacteria Improved by SynBio) such as insulin, growth hormones, and artemisinin production, and also on the food industry application with food coloring or artificial flavor production that already work.<br />
<br><br />
<br><br />
Of course, we have also presented our project as an innovative solution to kill and disperse biofilm in all pipelines or tanks and to prevent any colonization of bacteria.<br />
<br><br />
<br><br />
<br />
</div><br />
</div><br />
<h2>Part V: Public sensitization</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
To have an idea of how the public would react to the use of our solution in cleaning the installation for food and cosmetics industries, we have sent a survey in our school. We have received about 930 answers in less than a week, showing that people show interest in that subject.<br />
<br><br />
<br><br />
<h3>What are the public thoughts on the use of bacteria in cleaning process?</h3><br />
<br><br />
<br><br />
Studied population : <br />
<br><br />
<br><br />
<br />
We choose to interview a specific population : The larger part is constituted by scientific students who are from 18 to 24 years old .<br />
<br><br />
<br> <br />
<center><img src="https://static.igem.org/mediawiki/2012/a/ab/Age1.png" width="70%"/></center><br />
<br><br />
<br><br />
Moreover, their studies, knowledges or interests in Biology are different. The survey allows us to precise a tendency on the acceptance of bacteria in food and cosmetics industries by the young population, the future consumers. Furthermore, it gives us an idea whether our project would be workable and marketable.<br />
<br />
<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/de/Biology1.png" width="70%"/></center><br />
<br><br />
<br><br />
<br />
<br />
Results :<br />
<br><br />
<br><br />
First, we interest ourselves in the attention the public pays to the composition of the products they use in every day life.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c9/Concerned.png" width="70%"/></center><br />
<br><br />
<br><br />
In general, ¾ of the population is interested and concerned about what is put in their products. So, it seems relevant to ask them what they would accept or not.<br />
<br />
Foods and cosmetics are the products, people are more likely in contact with. Besides, they are industries we aim to clean with our bacteria.<br />
<br />
The important is to know the opinion of the larger public on this matter :<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/f/f5/Agecomparison.png" width="70%"/></center><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c5/Comparrsoncosm.png" width="70%"/></center><br />
<br><br />
<br><br />
remark : the bacteria are indicated as eliminated after cleaning and never in direct contact with the product<br />
<br><br />
<br><br />
<br />
As we could expect, the biological substances are the one people are more willing to accept, both in food and cosmetics industry.<br />
<br><br />
<br><br />
<br />
Moreover, There are not real concern for he use of living bacteria in these two fields.<br />
<br><br />
<br><br />
The questions of the chemicals and the GMO’s is quiet different : they are more accepted in cosmetics than in food (in general people seems more precocious on what they eat..) but generally less well see.<br />
<br><br />
<br><br />
The most remarkable is that the interviewed people have almost the same “reluctance” for the use of chemicals and GMOs. That can be interpreted as the evolution of the GMO’s status among students or their sensitization of chemicals’ effects.<br />
<br><br />
<br><br />
<br />
Some of the surveyed people belong to the 0-17 years old age group, and few to the more than 35 years old. The analysis of their answers leads us to note that the group above 35 years old has a tendency to refuse GMOs and to accept more easily living bacteria and chemicals. In the contrary, the youngest are more reluctant about chemicals in their foods , and least cautious on what are in their cosmetics.<br />
<br><br />
<br><br />
Through this survey, we remark no real issue to the public acceptance of Biofilm Killer in food and cosmetics industries. In fact, thanks to these results, we even feel an inducement in our search to limit the use of chemicals. <br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="introduction contenuTexte" style="margin:20px;font-size:15px;"><br />
<p><br />
<em> Special thanks to our faculty advisor O. Brette for his advice and for his working late to help us and to the UBC team for sharing its poll results with ours. </em><br />
</p><br />
<br><br />
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<div class="introduction contenuTexte" style="margin:20px;font-size:15px;margin-top:0px;"><br />
<p>Our human practice project includes two themes addressing many very different questions.<br />
The first ones are about various intellectual property issues. The other theme relates to the popularization of sciences and more specifically to our contribution to the researchers’ night.</p><br />
<p>We have chosen to divide the theme "intellectual property" into two parts because this theme is the main part of our reflexion and it required a longer development, but also because we collaborated with the <a href="https://2012.igem.org/Team:British_Columbia">University of British Columbia</a> on this theme and an entire part seemed necessary to explain our work.<br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and is made up by a reflexion on the economic challenges the synthetic biology industry faces. The second one sums up our collaborative work on the intellectual property with the UBC iGEM team. And the third one quickly presents our scientific popularization involvement. </p><br><br />
<br><br />
<br />
<h2>Part I: Intellectual property issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br />
Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements:iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
<br><br />
<br><br />
Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
<br><br />
<br />
The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Common's system", which will be discussed further on. We will assume the iGEM team members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:70%;"><br />
We will most notably refer to both Joseph Stiglitz’s (Nobel prize in economics in 2001) and Elinor Ostrom’s (Nobel prize in economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
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<h3>The patent system: an efficient economic model ?</h3><br />
<br />
<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first one to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
<br><br />
Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted invention have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
<br><br />
A patent is granted:<br><br />
<ul><br />
<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
</ul><br />
<br><br />
<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
<br><br />
The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even the genetically modified bacterias may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 80s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy.).<br><br />
<br> <br />
The goal of this brief study though, is not to discuss this matter.<br><br />
<br><br />
<div class="petitSsTitre">Why patents have been created?</div><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new age for intellectual property began.<br><br />
<br><br />
<br><br />
Instead, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
</div><br />
<br><br />
<br><br />
Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patent’s creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
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<br />
<br/><br />
As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
<br><br />
<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
<br><br />
<br><br />
For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components.When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
<br><br />
Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
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<div class="introduction contenuTexte" style="display:inline-block;width:65%;"><br />
As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licences of many expensive patents from the incumbent firms.<br><br />
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<br><br />
Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief resume. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ?. Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such, e.g., as shown in inventions and discoveries of more and better ways and means, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
</div><br />
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This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
<br><br />
Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing.In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
<br><br />
<div style="margin:30px;"><br />
<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
</div><br />
<br><br />
<br><br />
<br />
In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation.The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to embark on a collective reflexion on its contribution to the development of a "synthetic biology commonns”.<br />
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<h3>The Commons: an alternative model</h3><br />
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<br><br />
<br />
The open source software's users have been in ever greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies 92% used open source software during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
<br><br />
<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license.This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
<br />
<br><br />
<br><br />
The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
<br><br />
<br><br />
However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel prize in economics in 2009, broadened the economists' mind on this particular topic.<br />
<br><br />
<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th century.<br />
<br><br />
<br><br />
Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches: each community managing a commons fixes its own rules by defining a governance frameworkl and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
<br />
<br><br />
<br><br />
Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits.<br />
<br />
<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
<br />
<li>rules in use are well matched to local needs and conditions</li><br />
<br />
<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
<br />
<li>the right of community members to devise their own rules is respected by external authorities</li><br />
<br />
<li>a system for self-monitoring members’ behavior has been established</li> <br />
<br />
<li>a graduated system of sanctions is available</li> <br />
<br />
<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
<br><br />
Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
<br><br />
The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic value of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
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<br><br />
<br />
<h3>Bibliography</h3><br />
<br><br />
<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
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</div><br />
</div><br />
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<h2>Part II: FAQ</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br><br />
Our adventure in Amsterdam made us realize how abstract our reflexion was. We decided then to deepen it in a more concrete way through this FAQ which was inspired by the very questions we were asked in Holland.<br />
<br><br />
<br><br />
<i>Is it entirely impossible to patent an invention based on a public Part ?</i><br />
<br><br />
<br><br />
First of all, we shall remind the patent granting requirements :<br />
<br><br />
<br><br />
<li>the novelty condition (i.e. the invention must be partially or totally new);</li><br />
<li>the non-obviousness condition (U.S. patent law) or the inventiveness condition (in European patent law);</li><br />
<li>the usefulness condition (U.S. patent law) or the industrial applicability condition (in European patent law).</li><br />
<br><br />
<br />
<br />
Obviously, patenting public parts is impossible considering the first condition. However novel devices using public parts may be considered for protection through patents, depending on the degree of novelty carried by this device<br />
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<br><br />
<br />
<i>Could private actors like companies belong to and participate to a “synthetic biology commons” ?</i><br><br><br />
<br />
Fact : companies can take part in a commons. Many companies (Small and Medium Enterprises as well as big companies) currently do make profit in the open source software industry. This opportunity is not confined to the software industry: the idea of an “open source biology” has also emerged. This movement, also referred to as “open access biology”, has appeared in bio-industries such as the pharmaceutical sector, in most cases to prevent other companies to patent their research. The SNP Consortium, founded in 1999, gathers 10 pharmaceutical companies which decided to release some of their data concerning the human genome in the public domain. A few other more recent examples, including Pfizer (2006), Novartis (2007) and Syngenta (2002), tend to show that sharing knowledge has become part of the economic strategies in biology (Henkel & Maurer, 2007).<br><br />
<br><br />
Besides, such a sharing knowledge strategy allows companies to make good use of free resources, the validity of which has already been controlled by a (potentially) large community. Moreover, many business models may be designed (and have already been implemented to some extent), which are based on indirect profits, such as selling services for example.<br><br />
<br><br />
More generally, the ability of a commons to create value mainly derives from its capacity to link together different kinds of actors (companies, scholars, hackers, etc.) pursuing different objectives : making profits, publishing, or more original motivations, such as "the fun of programming", "the sense of belonging to a common culture, where participants share a common ideology, often characterized by reciprocity" (Henry and Stiglitz, 2010).<br />
<br><br />
<br><br />
<i>Can a commons coexist with other forms of the organization of economic activities (such as the market or the state involvement) inside the same sector ?</i><br />
<br><br />
<br><br />
Nothing opposes the possibility that different modes of coordination co-exist within the same sector. A given firm may share knowledge in a commons perspective, while simultaneously developing market relations. For instance, some software vendors sell proprietary software (“proprietary bricks”) that complement a main open source program (which one is free to use) like an option.<br><br />
<br><br />
Existing economic associations such as the BiOS (“Biological innovation for an Open Society”) Initiative of Cambia, suggest that such a combination would be possible in the field of synthetic biology. The BiOS license allows the licensees to use Cambia enabling technology (a few key plant gene transfer patented technologies) royalty-free, on condition that the improvements made to the technology are also made freely available (i.e. they can be used royalty-free by other licensees). Furthermore, the BiOS founders, who claim that they have adapted the “open source” approach to biology (the source code being here the enabling technology), also argue that their licensing device does not lessen the usual incentives to innovate, including the possibility of patenting some products developed from the application of the enabling technology. <br><br />
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<br />
<br />
<i>Why try to find our own rules for the iGEM commons ? The open source seems to work great, so shouldn’t we use theirs ?</i><br />
<br><br />
<br><br />
Ostrom put forward a set of generic principles for a commons to be economically viable. One of them is that every single commons has its own rules that are defined by both its actors and the resources that are dealt with.<br />
Not only are the actors of the open source software movement different from the synthetic biology community but also the code source is a resource different from the biological parts.These reasons make it hardly possible that the same rules could be applied to both commons, especially on the legal point of view. Indeed, the open source software movement is based on the spreading of open source licenses, which derive from copyright (“copyleft”). Such a solution is generally considered by legal experts as unadapted to biological parts (Henkel & Maurer, 2007). These experts also suggest that an economic model combining patents with legal devices ensuring free knowledge sharing and use would be the best way to the economic development of synthetic biology and to the spreading of innovations.<br />
<br><br />
<br><br />
<br />
<i>Should not we consider that the bases of a synthetic biology commons have already been defined, by several organisms, including the iGEM Foundation itself ?</i><br />
<br><br />
<br><br />
<br />
The iGEM community has already some attributes of a commons. However, we consider that this commons could be better structured and could play a far bigger role in orienting the development of synthetic biology.<br />
The objectives of our initiative are threefold :<br />
<br><br />
<br><br />
<li>first, leading every member of the iGEM community to be aware of participating to a commons;</li><br><br />
<li>second, provoking a debate within the iGEM community to precise its founding rules. The community should collectively deliberate on the definition of a set of rules regarding the rights to access BioBricks, to use them, to control their application, to impose sanctions if necessary, etc.;</li><br><br />
<li>third, leading the iGEM community to discuss its place and the role it may play in the development of an overall synthetic biology commons, involving different kinds of actors : other associations such as the BiOS Initiative, public research units and higher education organizations, companies, and so on. Though building an overall synthetic biology commons is far from being an easy task, it seems to be a growing concern for many actors, including public authorities. For instance, the current Minister of Higher Education and Research in France, Geneviève Fioraso, recently wrote a comprehensive Parliamentary Report on the scientific, technological and economic stakes of synthetic biology, which was published a few months before becoming a Minister (Fioraso Report, 2012). This report notably highlights the crucial role of iGEM in the development of synthetic biology.</li><br><br />
<br />
<br><br />
<br><br />
<h3>Bibliography</h3><br />
<br><br />
We used all the previous references (q.v. part I), though we added : <br><br />
<ul><br />
<li>Rai A, Boyle J (2007) <em>Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons.</em> PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058</li><br><br />
<li>Bios website : <a href="http://www.bios.net/daisy/bios/home.html">Bios website</a></li><br><br />
<li> Henkel J, Maurer S (2007) <em>The economics of synthetic biology</em>. Mol Syst Biol 3: 117. </li><br><br />
<li> Fioraso G (2012) <em>Les enjeux de la biologie de synthèse</em>. Report for the "Office parlementaire d'évaluation des choix scientifiques et technologiques".<br />
</ul><br />
<br><br><br />
</div><br />
</div><br />
<br />
<h2>Part III: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey.281 iGEMers had answered the survey when we treated it .<br />
<br><br />
This collaboration allowed us to broaden our reflexion to iGEM teams' issues.<br />
<br><br />
<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly we studied past iGEMers experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
<br><br />
Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
<br><br />
Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may results in a patent experience if the materials used are under a proprietary license. <br />
<br><br />
Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers IP experiences.<br />
<br/><br />
<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what was its nature. The results are that most of them (46 %) had already used patented materials before. However, 15% renounced to actually use them, which shows how patent can impede research and innovation: getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
<br><br />
The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10 % of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
<br><br />
Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
<br><br />
Our sole conclusion on this matter is that the IPR often does affect iGEM projects, even if in general the patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
<br />
<br><br />
It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
<br><br />
Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
<br><br />
To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member or / and to a faculty advisor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
<br><br />
<br><br />
<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the intellectual property rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks .<br />
<br><br />
Besides, this survey deepened our main reflexion (q.v. part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the intellectual property rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
</div><br />
</div><br />
<br />
<h2>Part IV: Popularization of Science</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
Stereotypes and various pieces of information about science are spread in such a way that people sometimes find it hard to decipher the truth about everything that is said. The public, but also non-biologist scientists, may sometimes have to look for a reliable source to make their own opinion. <br><br />
We are convinced that we have a role to play and have decided to involve ourselves in several actions.<br />
<br><br />
<br><br />
<h3>First, we organized three conferences with cutting-edge experts:</h3><br />
<ul><br />
<li>“Bacterial swimmers can penetrate biofilms, making them vulnerable to destruction.” by Romain Briandet, expert in Surface Hygiene/Bioadhesion in May 2012.<br />
This meeting enabled the Lyon-INSA iGEM team to discover the amazing properties of Bacillus subtilis swimmers and their potential as a tool for biofilm control.</li><br />
<li>“Synthetic Biology : Biotechnologies revival?” by François Képès, national and international SynBio expert in September 2012. Képès initiated a debate on a paradox that biotechnologies raise. Paradoxically, biotechnologies aim at improving the industrialization of their products with normalization, but also intend to be more creative by setting free from existing constraints.</li><br />
<li>Olivier Brette in September 2012. The stakes of intellectual property, science and innovation were presented and discussed with the assembly of students and staff from various scientific fields (mechanics, informatics, economic intelligence, ethics…).</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Then, we organized meetings with non-biologist scientists and the public.</h3><br />
<ul><br />
<li>From May to September 2012 : Open debates on the stakes of SynBio with staff and students from Mechanics, Informatics and Telecoms INSA departments but also staff from INSAVALOR (Research and Development, Research Valuation of INSA-affiliated company) through the “Filière Ingénieur-Entreprendre” (Entrepreneurship formation). Participation to the iGEM Entrepreneurship Division was even considered.</li><br />
<li>September 2012 : The Researchers' Night. Since 7 years, this event has been taking place on a single September night in about 300 cities all over Europe. The main goal of this night is to put researchers in touch with the public. Thus, they can explain what they are doing, how and what can be the applications in the day-to-day life. <br />
<br><br />
This year, the theme was "Imagine the future": what future will emerge from research laboratories? How do scientists imagine the future? Archeology, Physics, Philosophy, Biosciences or Linguistic: researchers shared their lab experiences and thoughts on the future. Therefore, in order to defend a controversial discipline, Synthetic Biology (a.k.a. SynBio), the Lyon-INSA team members participated in the Researchers’ Night, which was held on September 28th, 2012, at the CCO of Villeurbanne.<br />
<br><br />
Indeed, an article published by a French team: “Séralini et al., Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize, Food and Chemical Toxicology, Volume 50, Issue 11, November 2012, Pages 4221–4231” and also in the paper “Le Monde” on September 25th, 2012, on deleterious effect of transgenic maize on rats, had a dramatic effect on the public opinion in Europe. Thus, to illustrate that SynBio can be used to improve the environment, health and life of people, we decided to explain the potential of SynBio and to exchange with the public through a scientific speed-dating and around a table containing diagrams and Petri dishes.<br />
<br><br />
“What is it?” asked a little boy. “What are you doing?” asked a woman. We could introduce them to our friends: bacteria! Then, one of the iGEM team members answered: “Bacteria are little organisms which grow, eat and die like every living being. These bacteria are not always pathogenic and scientists can improve their qualities with the introduction of DNA fragments containing genes.”<br />
<br><br />
So the objective is reached: we aroused the surprise, the questioning and the amazement of people. In this respect, we pointed out the usefulness of SynBio. We decided to focus on medical application of BABS (Bacteria Improved by SynBio) such as insulin, growth hormones, and artemisinin production, and also on the food industry application with food coloring or artificial flavor production that already work.<br />
<br><br />
Of course, we have also presented our project as an innovative solution to kill and disperse biofilm in all pipelines or tanks and to prevent any colonization of bacteria.<br />
<br><br />
</li><br />
</ul><br />
<br />
<br />
</div><br />
</div><br />
<h2>Part V: Public sensitization</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
To have an idea of how the public would react to the use of our solution in cleaning the installation for food and cosmetics industries, we have sent a survey in our school. We have received about 930 answers in less than a week, showing that people show interest in that subject.<br />
<br><br />
<br><br />
<h3>What are the public thoughts on the use of bacteria in cleaning process?</h3><br />
<br><br />
<br><br />
Studied population : <br />
<br><br />
<br><br />
<br />
We choose to interview a specific population : The larger part is constituted by scientific students who are from 18 to 24 years old .<br />
<br><br />
<br> <br />
<center><img src="https://static.igem.org/mediawiki/2012/a/ab/Age1.png" width="70%"/></center><br />
<br><br />
<br><br />
Moreover, their studies, knowledges or interests in Biology are different. The survey allows us to precise a tendency on the acceptance of bacteria in food and cosmetics industries by the young population, the future consumers. Furthermore, it gives us an idea whether our project would be workable and marketable.<br />
<br />
<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/de/Biology1.png" width="70%"/></center><br />
<br><br />
<br><br />
<br />
<br />
Results :<br />
<br><br />
<br><br />
First, we interest ourselves in the attention the public pays to the composition of the products they use in every day life.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c9/Concerned.png" width="70%"/></center><br />
<br><br />
<br><br />
In general, ¾ of the population is interested and concerned about what is put in their products. So, it seems relevant to ask them what they would accept or not.<br />
<br />
Foods and cosmetics are the products, people are more likely in contact with. Besides, they are industries we aim to clean with our bacteria.<br />
<br />
The important is to know the opinion of the larger public on this matter :<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/f/f5/Agecomparison.png" width="70%"/></center><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/c5/Comparrsoncosm.png" width="70%"/></center><br />
<br><br />
<br><br />
remark : the bacteria are indicated as eliminated after cleaning and never in direct contact with the product<br />
<br><br />
<br><br />
<br />
As we could expect, the biological substances are the one people are more willing to accept, both in food and cosmetics industry.<br />
<br><br />
<br><br />
<br />
Moreover, There are not real concern for he use of living bacteria in these two fields.<br />
<br><br />
<br><br />
The questions of the chemicals and the GMO’s is quiet different : they are more accepted in cosmetics than in food (in general people seems more precocious on what they eat..) but generally less well see.<br />
<br><br />
<br><br />
The most remarkable is that the interviewed people have almost the same “reluctance” for the use of chemicals and GMOs. That can be interpreted as the evolution of the GMO’s status among students or their sensitization of chemicals’ effects.<br />
<br><br />
<br><br />
<br />
Some of the surveyed people belong to the 0-17 years old age group, and few to the more than 35 years old. The analysis of their answers leads us to note that the group above 35 years old has a tendency to refuse GMOs and to accept more easily living bacteria and chemicals. In the contrary, the youngest are more reluctant about chemicals in their foods , and least cautious on what are in their cosmetics.<br />
<br><br />
<br><br />
Through this survey, we remark no real issue to the public acceptance of Biofilm Killer in food and cosmetics industries. In fact, thanks to these results, we even feel an inducement in our search to limit the use of chemicals. <br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="introduction contenuTexte" style="margin:20px;font-size:15px;"><br />
<p><br />
<em> Special thanks to our faculty advisor O. Brette for his advice and for his working late to help us and to the UBC team for sharing its poll results with ours. </em><br />
</p><br />
<br><br />
</div><br />
<br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/notebookTeam:Lyon-INSA/notebook2012-10-25T20:08:58Z<p>Suxiaohui: </p>
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<project><br />
<month name="July" size="31"><br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 2nd 2012</date><br />
<description>Liquid culture (5 mL LB medium) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description> <br />
</jour><br />
<jour nb="3"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 3rd 2012</date><br />
<description><p>Dilution of 100 µL saturated culture in 5 mL LB medium. </p><br /><br />
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 0.3). </p><br /><br />
Transformation of the NM522 strain (this experiment was repeated 3 times). <br />
<ul><br />
<li>For the positive control the pSB1C3 plasmid was used;</li><br />
<li>For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).</li><br />
</ul><br />
The transformed bacteria were selected on chloramphenicol (Cm) plates.<br />
</description><br />
</jour><br />
<jour nb="4"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 4th 2012</date><br />
<description><br />
Transformation analysis :<br/><br/><br />
<ul><br />
<li>Positive control : lots of colonies;</li><br />
<br />
<li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the two other negative control plates;</li><br />
<br />
<li>Test plate : between 1 and 8 were observed.</li></ul><br />
<br/><br />
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/><br />
<br />
The antibiotic resistance to Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin of the following strains was tested : BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS <i>abrB</i>.<br />
</description><br />
</jour> <br />
<jour nb="5"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 5th 2012</date><br />
<description><br />
Antibiotics resistance tests :<br/><br/><br />
<ul><br />
<li>Bs 168 : no resistance;</li><br />
<li>Bs 168 M cherry : no resistance;</li><br />
<li>Bs 168 GFP : no resistance;</li><br />
<li>Bs <i>abrB</i> : Cm resistant;</li><br />
<li>Bs 168 lysostaphin PWG100 : no resistance;</li><br />
<li><i>S. epidermidis</i> : Tet resistant.</li><br />
</ul><br />
<br/><br />
Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after <i>Eco</i>RI digestion)<br />
<br />
</description><br />
</jour><br />
<jour nb="6"><br />
<titre>For all purposes</titre><br />
<date> Friday, July 6th 2012</date><br />
<description><br />
<br />
Phone conference with Romain Briandet.<br/><br/><br />
<br />
Terminator was retrieved from plate 1 well 13D.<br/><br/><br />
Long meeting.<br />
<br />
</description><br />
</jour><br />
<jour nb="9"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 9th 2012</date><br />
<description><br />
<ul><br />
<li>pBBa_I742123 was put in storage (under the reference pBK1).</li><br />
<li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB medium + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li><br />
<li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li><br />
<li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li><br />
<li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</li><br />
<li>Liquid cultures of the following strains were made : Bs 168 in LB, Bs <i>abrB</i> in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul><br />
<br />
</description><br />
</jour><br />
<jour nb="10"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 10th 2012</date><br />
<description><br />
<ul><br />
<li> The following parts were put in storage :</li><br />
<ul><br />
<li>Lysostaphin in pUC57 Amp resistant (pBK2);</li><br />
<li>Dispersin in pUC57 Amp resistant (pBK3);</li><br />
<li>Surfactin part 2 (RBS-<i>lacI</i>-terminator) in pUC57 Amp resistant (pBK4).</li><br />
</ul><br />
<li> and the following strains (in LB broth supplemented with required antibiotic) :</li><br />
<ul> <br />
<li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li><br />
<li>Bs <i>abrB</i> = BK2 (Cm<sup>R</sup>);</li><br />
<li>Bs 168 = BK3.</li><br />
</ul><br />
<li>Ligation of Dispersin and Lysostaphin biobricks :</li><br />
<ul><br />
<li>digestion of pBK2 with the restriction enzymes <i>Eco</i>RI and <i>SpeI</i>;</li><br />
<li>digestion of pBK3 with the restriction enzymes <i>PstI</i> and <i>XbaI</i>;</li><br />
<li>digestion of pBK4 with the restriction enzymes <i>EcoRI</i> and <i>PstI</i>;</li><br />
<li>3A ligation of the digested parts;</li><br />
<li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2.1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.</li></ul><br />
<li>Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 bands are observed : <strong>PROBLEM. Digestion is made again with E, P and E+P → There are 2 <i>PstI</i> sites !!! WRONG PLASMID</strong></li><br />
<br />
<li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li><br />
<li>Design and order of the primers for the constitutive promoter (part BBa_K143012).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 11th 2012</date><br />
<description><br />
<ul><br />
<li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain.</li><br />
<li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain. <i>(NB : the clones were pink which means that the part contains a RFP gene)</i></li><br />
<li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain.</li><br />
<li>Extraction of pUC57-Lysostaphin/pUC57-Dispersin/pUC57-<i>lacI</i> from transformed NM522 strains.</li><br />
<li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the color of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 12th 2012</date><br />
<description><br />
<ul><br />
<li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li><br />
<li>Reception and storage of the primers (for the amplification of the BBa_K143012 part).</li><br />
<li>The transformed NM522 strain with the part BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit.</li><br />
<li>Extractions with a miniprep kit are made :<br />
<ul><br />
<li>4 clones containing the P<sub>lac</sub> promoter (1,2,3,4);</li><br />
<li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li><br />
<li>3 clones containing theTerminator (1,2,3);</li><br />
<li>4 clones containing the gene for Dispersin;</li><br />
<li>4 clones containing the gene for Lysostaphin;</li><br />
<li>4 clones containing the gene for <i>lac</i>I.</li><br />
</ul><br />
Then a digestion is made on a 0.7% agarose gel.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<titre>Kill</titre><br />
<date> Friday, July 13th 2012</date><br />
<description><br />
<ul><br />
<li>PCR of the constitutive promoter (part BBa_K143012) → the PCR did not work so we ran a new PCR with a more diluted promoter solution.</li><br />
<li>The following strains are put in storage:<br />
<ul><br />
<li>NM522 containing <i>lacI</i>-pUC57;</li><br />
<li>NM522 containing RBS-pUC57;</li><br />
<li>NM522 containing dispersin-pUC57;</li><br />
</ul><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date> Monday, July 16th 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
<ul><br />
<li>PCR of constitutive promoter → it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li><br />
<li>Purification of the PCR product.</li><br />
<li>Gel electrophoresis of lysostaphin.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation of B0015 terminator.</li><br />
<li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date> Tuesday, July 17th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Long morning meeting to discuss results and to write some posters in a frenzied psychopath way in order to remember our tasks.</li><br />
<li>Ligation of promoter-RBS-<i>gfp</i> in plasmid.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168: buffers preparation.</li><br />
<li>Failure of B0015 transformation.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/<i>gfp</i> (of <i>E. coli</i>) using a 3A ligation followed by transformation of the ligation in the NM522 strain;</li><br />
<li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> in 5mL of TSB (conditions described in protocol “Tests on Staphylococcus aureus biofilms in 24 well plate” ).</li><br />
<li>A bacterial suspension of <i>S. epidermidis</i> with OD<sub>600</sub> close to 0.132 is prepared from a Petri dish containing <i>S. epidermidis</i>. A microtiter plate is inoculated with 2 mL of the suspension diluted 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="18"><br />
<date> Wednesday, July 18th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid → extraction failure.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168 (Kit Genomic DNA from tissue).</li><br />
<li>Transformation of pBK5 in <i>B. subtilis</i> <i>abrB</i> failed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation results of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;</li><br />
<li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis : there is still a smear. However, the bands are as expected.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i>.</li><br />
<li>A microtiter plate is inoculated with 2mL of a suspension obtained from diluting the liquid culture 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="19"><br />
<date> Thursday, July 19th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work.</li><br />
<li>A 50µg/mL erythromycin solution is made in order to test the strains’ resistance;</li><br />
<li>Transformation of <i>yfp</i>, <i>gfp</i> and <i>cfp</i> (from iGEM plates) in NM522;</li><br />
<li>B0015 transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth supplemented with Tetracyclin.</li><br />
<li>The fluorescence test of the transformed bacteria containing [dispersin+RBS/<i>gfp</i>+pSB1T3] confirm that the promoter is functional.</li><br />
<li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i> and that the fact of cultivating the strain in broth or on solid medium has no impact on the biofilm's quality.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date> Friday, July 20th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and promoter+pSB1C3 OK → 6 clones are isolated on a LB+Cm plate.</li><br />
<li>Quantification and gel electrophoresis of <i>B. subtilis</i> genomic DNA.</li><br />
<li>Antibiotic testing of <i>B. thuringensis</i> 407 <i>gfp</i> strain and other strains in LB+Ery growth medium.</li><br />
<li>Failure of transforming pBK5 in <i>B. subtilis</i> 168 with the same protocol as previously. New task : find a new protocol.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin.</li><br />
<li>Ligation Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.</li><br />
<li>Ligation Lysostaphin in pSB1C3 and transformation.</li><br />
<li>Isolation of 6 clones of the Terminator transformation.</li><br />
<li>Transformation results of fluorescent genes : ok, except <i>yfp</i>. </li><br />
<li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date> Monday, July 23rd 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Selected clones (NM522+pBK6) are red, meaning that P<sub>veg</sub> promoter and RBS from <i>B. subtilis</i> are recognized by <i>E. coli</i>’s ribosome. 4 clones are streaked in LB+Cm.</li><br />
<li>LB+Ery and TSB+Tet Petri plates are made.</li><br />
<li><i>yfp</i> transformation was successful.</li><br />
<li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li><br />
<li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li><br />
<li>Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results</li><br />
<ul><br />
<li>Promoter+Dispersin in pIG23 : nothing on the plate;</li><br />
<li>Lysostaphin, negative control contains bacteria so the plate is put in junk.</li><br />
</ul><br />
<li>New digestions, ligations,transformations:</li><br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57;</li><br />
<li>Dispersin in pSB1C3;</li><br />
<li>Lysostaphin in pSB1C3;</li><br />
<li>Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).</li><br />
</ul><br />
<li>Streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promoter and the RBS specific to the <i>Bacillus subtilis</i> strain are recognized by the RNA polymerase of <i>E. coli</i>.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR of xylR. Gel electrophoresis : the PCR didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date> Tuesday, July 24th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.</li><br />
<li>Extraction of <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ; test → OK : put in storage.</li><br />
<li>Extraction of the pHT315 GFP plasmid of <i>B. thuringiensis</i> 407 GFP to build a shuttle vector <i>B. subtilis</i> ↔ <i>E. coli</i>. Transformation in NM522 strain and selection on LB+Ampicillin medium : ok.</li><br />
<li><i>S. epidermidis</i> is put in storage in TSB medium under the reference BK15.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results:<br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;</li><br />
<li>Promoter+Dispersin : nothing on the plate;</li><br />
<li>Dispersin in pSB1C3 : a lot of clones;</li><br />
<li>Lysostaphin in pSB1C3 : a lot of clones.</li><br />
</ul><br />
Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (because there are too many clones!)</li><br />
<br />
<li>Extraction of the plasmidic DNA [Promoter in pSB1C3].</li><br />
<li>Extraction of pBK6 from the transformed strain.</li><br />
<li>Extraction of the [Promoter+RBS+<i>gfp</i>] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by <i>Eco</i>RI and <i>Pst</i>I : gel ok. Congrats (pBK12) → 122,6 ng/µL.</li><br />
<li>Extraction of the pBK6 plasmid (P<sub>veg</sub>+RBS+RFP in pSB1C3). The electrophoresis confirms the plasmid’s structure.</li><br />
<li>Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New PCR of xylR with some modifications in the protocol : the PCR didn’t work.<br />
<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date> Wednesday, July 25th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Strains are put in storage :</li><br />
<ul><br />
<li><i>S. epidermidis</i> in TSB+Tet (BK17);</li><br />
<li><i>B. thuringensis</i> 407 GFP in LB+Ery (BK16).</li><br />
</ul><br />
<li>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).</li><br />
<li>Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (GFP or not).</li><br />
<li>The plasmid extraction protocol from <i>B. subtilis</i> was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of the extracted plasmidic DNA of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.</li><br />
<li>Selection of 4 clones transformed by [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.</li><br />
<li>Miniprep to extract [Lysostaphin+Cm] and [Dispersin+Cm] → Digestion.</li><br />
<li>3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).</li><br />
<li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on LB+Tet plates having a biofilm already established and in development of <i>S. epidermidis.</i></li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR on the <i>B. subtilis</i> 168 genome using universal primers of Phil Oger, and positive control with <i>Bulkhoderia cepacia</i> genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of <i>B. subtilis</i> 168. <br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date> Thursday, July 26th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for <i>B. subtilis</i> and <i>E. coli</i>).</li><br />
<li>gDNA extraction with 2 different protocols : the first for <i>Bacillus subtilis</i> 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.</li><br />
<li>PCR of rRNA 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.</li><br />
<li>NM522 + <i>gfp</i>, <i>cfp</i> and <i>yfp</i> in storage.</li><br />
<li>pHT315 GFP put in storage under the reference pBK18.</li><br />
<li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other verification by extracting their plasmidic DNA (but they are not good again).</li><br />
<li>Extraction of the plasmidic DNA of the clones transformed with Lysostaphin+Dispersin in pBK2 and verification by electrophoresis gel. The clones are not right.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A assembly RBS-<i>gfp</i> (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date> Friday, July 27th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
Extraction of transformed clones (NM522/pHT304 and NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites <i>Xba</i>I, <i>Eco</i>RI and <i>Pst</i>I as expected. However, there is a <i>Spe</i>I site which had not been mapped.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.</li><br />
<li>Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.</li><br />
<li>New 3A ligation with Lysostaphin/Terminator/pSB1C3 and transformation in NM522 strain.</li><br />
<li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 and pBK17.</li><br />
<li>Failure of transformation of L1 in NM522.</li><br />
<li>3A assembly of RBS-<i>cfp</i> (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li><br />
<li>3A assembly of <i>sfp</i>-part2(<i>lacI</i>)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>PCR of <i>xylR</i> from gDNA extracted yesterday. Test on gel : successful PCR !!</li><br />
<li>3A ligation of RBS (pBK7) and <i>xylR</i> (produced by PCR) in pSB1C3 ( ! the vector plasmid has the same resistance as the plasmid containing the RBS...).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date> Monday, July 30th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li><strong>It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire…).</strong></li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion test and gel of pBK2 (promoter + lysostaphin) by <i>Eco</i>RI and <i>Spe</i>I, pBK3 (dispersin + terminator) by <i>Xba</i>I and <i>Pst</i>I and pSB1C3 by <i>Eco</i>RI and <i>Pst</i>I.</li><br />
<li>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel → the 3 clones aren’t right.</li><br />
<li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD<sub>600</sub> = 0.5, dilution the culture x100, 1000 and 10 000).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of L1, L2 and IV in NM522.</li><br />
<li>Transformation of <i>sfp</i> and <i>abrB</i> in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Purification of <i>xylR</i>, produced by PCR.<br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date> Tuesday, July 31st 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin).<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i><i>sfp</i></i> parts.</li><br />
<li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of RBS and <i>xylR</i> and transformation in NM522 strain.<br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="August" size="31"><br />
<jour nb="1"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, August 1st 2012</date><br />
<description><br />
<br />
<p>The transformation protocol for <i>Bacillus</i> was tested using the pHT315 GFP plasmid extracted from the <i>B. thuringiensis</i> BT407GFP strain.</p><br />
<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>Lysostaphin+terminator : nothing;</li><br />
<li>Lysostaphin+dispersin : a lot of clones.</li><br />
</ul><br />
Cultures in LB broth of Lysostaphin+Dispersin are launched.</li><br />
<li>Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.</li><br />
<li>pUC57 with Dispersin put in storage : pBK3.</li><br />
<li>Lysostaphin test returns ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The strain NM522/pBK6 was put in storage under the reference BK22.</li><br />
<li>Transformations of the NM522 strain with the ordered constructions (<i>sfp</i> and <i>abrB</i> in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li><br />
<li>Quantification of <i>sfp</i> and <i>abrB</i> constructions provided by Genecust using the Nanodrop.</li><br />
<li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li><br />
</ul><br />
</description> <br />
</jour><br />
<br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Thursday, August 2nd 2012</date><br />
<description><br />
<ul><br />
<li>The transformation of the <i>Bacillus</i> strain failed because of contaminated LB broth, so a new transformation was attempted.</li><br />
<li>pHT304 and pHT315 plasmids supernumerary site <i>Spe</i>I deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dispersin clones digestion : clones not okay.</li><br />
<li>Ligations of :<br />
<ul><br />
<li>Constitutive promoter in Cm iGEM plasmid;</li><br />
<li>Dispersin in Cm iGEM plasmid.</li><br />
</ul><br />
Ligation were verified by electrophoresis.</li><br />
<li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the <i>Bacillus</i> colonies where <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li><br />
<li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the <i>abrB</i> construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li><br />
<li>New ligation of RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <I>sfp</i> and <i>abrB</i> in NM522 strain didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date> Friday, August 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Purification of the NM522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li><br />
<li>The results of <i>Bacillus</i> transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li><br />
<li>A lot of clones for the transformation of the pHT304 and pHT315 plasmids (without <i>Spe</i>I site) on LB+Amp plates → Purification of 12 clones each.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li><br />
<li>Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).</li><br />
<li>BK12 strain verification : spreading on LB + Amp plate.</li><br />
<li>Ligation of Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li><br />
<li>Ligation were verified by electrophoresis.</li><br />
<li>Transformation of Promoter+Dispersin ligation.</li><br />
<li>PCR to check the presence of the promoter in the Promoter in Cm iGEM plasmid clones.</li><br />
<li>Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill <i>Bacillus</i>, 500 µL of Bs 168 pWG100 supernatant, 500 µL of <i>S. epidermidis</i> with OD<sub>600</sub>=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD<sub>600</sub> with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+<i>cfp</i>+pSB1K3).</li><br />
<li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+<i>gfp</i>+pSB1K3).</li><br />
<li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li><br />
<li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New ligation between RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date> Saturday, August 4th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>The negative control is ok;</li><br />
<li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li><br />
</ul><br />
8 clones for each transformation are chosen and spread on LB+Cm and LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformations of <i>abrB</i> and <i>sfp</i> are a success !! :D</li><br />
<li>Liquid cultures of <i>abrB</i> and <i>sfp</i> clones are launched to do plasmid extractions.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Sorting of the RBS + <i>xylR</i> clones to eliminate the clones containing two relegated plasmids.<br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date> Sunday, August 5th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 S.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Plasmid extractions from 3 clones NM522/<i>abrB</i> and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp).<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Liquid cultures of RBS+<i>xylR</i> clones are launched to do plasmid extractions.<br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date> Monday, August 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Plasmid extraction from 6 clones Bs 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li><br />
<li>Miniprep and digestions by <i>Spe</i>I of the plasmid extracted from the 6 clones of NM522 with pHT304 S and NM522 with pHT315 S : Failure ! The <i>Spe</i>I site is still in the plasmids :’( </li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Selection of clones growing on LB+Cm plates and not on LB+Amp plates :<br />
<ul><br />
<li>4 clones Dispersin in pSB1C3;</li><br />
<li>7 clones Promoter pSB1C3;</li><br />
<li>5 clones Promoter+Dispersin in pSB1C3.</li><br />
</ul><br />
Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li><br />
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on LB+Cm and LB+Amp to test their resistance.</li><br />
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of the following ligations, in NM522 strain :</li><br />
<ul><br />
<li>pBK7+pBK13+pSB1K3 (RBS+<i>cfp</i> in Kanamycin resistant backbone);</li><br />
<li>pBK7+pBK14+pSB1K3 (RBS+<i>gfp</i> in Kanamycin resistant backbone).</li><br />
</ul><br />
<li>PCR of RBS-<i>abrB</i> and P<sub>xyl</sub> and purification of these PCR products.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Purification of the <i>xylR</i> gene.</li><br />
<li>Miniprep of RBS-xylR and electrophoresis test → the ligation failed again...</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Tuesday, August 7th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>There is a little plasmid pHT304 S and pHT315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without <i>Spe</i>I site.</li><br />
<li>Transformation in NM522 and spreading on LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li><br />
<li>Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).</li><br />
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li><br />
<li>New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours at 37°C (efficiency of the lysostaphin in stationary phase ?) → the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>3A ligation : [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + [psB1T3].</li><br />
<li>Transformation of the ligation [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + pSB1T3 in NM522 strain and spreading LB+Tet plates.</li><br />
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li><br />
<li>Purification of P<sub>xyl</sub> produced by PCR.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Wednesday, August 8th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 by pHT304 S and pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the <i>Spe</i>I site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...</li><br />
<li>Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.</li><br />
<li>New ligations : <br />
<ul><br />
<li>Promoter+Dispersin in pSB1C3;</li><br />
<li>Lysostaphin+Dispersin in pSB1C3.</li><br />
</ul><br />
Then transformation in NM522.</li><br />
<li>Plasmidic extraction of the BK12 strain : plasmid okay.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Results of the transformations :<br />
<ul><br />
<li>Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li><br />
<li>Transformation by <i>sfp</i>-<i>abrB</i>-pSB1T3 : the negative control isn’t good, the transformation is done again...</li><br />
</ul><br />
<li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Thursday, August 9th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Extraction of NM522/pHT315S. We expected a plasmid without a SpeI site. However, the selected clone contained a plasmid identical to pHT315 (the digestion by <i>Spe</i>I and <i>Eco</i>RI enzymes gives a fragment at 1000 bp). We decided to screen more clones (8 out of 17).</li><br />
<li>The transformation pSB1C3 + RFP and pSB1T3 + RFP is a success ! (red clones)</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
Transformation results : <br />
<ul><br />
<li>Positive control : okay;</li><br />
<li>Negative control : okay;</li><br />
<li>[Lysostaphin + Dispersin] : 8 clones;</li><br />
<li>[Promoter + Dispersin] : more than 30 clones.</li><br />
</ul><br />
Clones are screened on LB + Amp and LB + Cm plates.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The transformation of NM522 strain by <i>sfp</i>-<i>abrB</i> didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the <i>sfp</i>-<i>abrB</i> ligation again...</li><br />
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li><br />
<li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Friday, August 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Deletion of the <i>Spe</i>I site from pHT315 and pHT304 plasmids and filling-in using the <i>Pfu</i> polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li><br />
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.</li><br />
<li>None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.</li><br />
<li>Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.</li><br />
<li>Plasmidic extractions of these clones : clones not okay.</li><br />
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.</li><br />
<li>The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li><br />
<li>The NM522 strain with <i>abrB</i>-pSB1K3 was put in storage under the reference BK25.</li><br />
<li>The NM522 strain with <i>sfp</i>-pSB1K3 was put in storage under the reference BK26.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Saturday, August 11th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Gel electrophoresis showed that the elimination of the <i>Spe</i>I site was not successful.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Sunday, August 12th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Culture of 60 mL of Bs 168 pWG 100 at 28°C for the lysostaphin tests on plates.<br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Monday, August 13th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>We identified the reason why the deletion of the site <i>Spe</i>I from pHT315 and pHT304 plasmids and filling-in was not successful : the <i>Pfu</i> enzyme requires Mg<sup>2+</sup> and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li><br />
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth. These flasks are then frozen at -80°C and freeze-dried overnight.</li><br />
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligation [<i>sfp</i> + <i>abrB</i> + pSB1A3] in the NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Tuesday, August 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The gel electrophoresis of the digested plasmids pHT315 and pHT304 after the final purification shows promising results, so the two plasmids were transformed in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li><br />
<li>Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformation of the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Standard ligation between pBK7 (=RBS in pSB1C3) and xylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain. <br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Wednesday, August 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.</li><br />
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
None of the 4 clones tested had the expected fragments, so we screened 6 others.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by pBK7 and <i>xylR</i> : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + <i>xylR</i> contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and <i>xylR</i> (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Thursday, August 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones having integrated the modified shuttle vector (after filling-in). 4 clones seem to have the right plasmid.</li><br />
<li>The electrophoresis after digestion with <I>Eco</i>RI and <i>Spe</i>I showed that the <i>Spe</i>I site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive promoter (P<sub>veg</sub>) produced by PCR in an iGEM plasmid.</li><br />
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).</li><br />
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.</li><br />
<li>Ligation of P<sub>xyl</sub> produced by PCR in an iGEM plasmid.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Ligation of <i>xylR</i> produced by PCR in an iGEM plasmid (pSB1K3).</li><br />
<li>Results of the transformation of NM522 by [pBK7 + <i>xylR</i>] (for the second ligation) : the plate with bacteria transformed by [pBK7 + <i>xylR</i>] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + <i>xylR</i>]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without <i>xylR</i>.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Friday, August 17th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time, the digestion was successful. The tests showed that there was no <i>SpeI</i> site. However, the pHT315 plasmid also had no XbaI site. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a <i>XbaI</i> site. The digestion of the control plasmid was partial.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Plasmid extractions from the 14 clones transformed by [pBK7 + <i>xylR</i>] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without <i>xylR</i>.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Monday, August 20th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>pBK19 with no SpeI site was put in storage under the reference pBK25.</li><br />
<li>pBK20 with no SpeI site was put in storage under the reference pBK26.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Launch of 8 [Promoter+Dispersin] cultures.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + P<sub>xyl</sub>] : the plate with bacteria transformed by [pSB1T3 + P<sub>xyl</sub>] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + RBS-<i>abrB</i>] : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by [pSB1K3 + <i>xylR</i>] : the plate with bacteria transformed by [pSB1K3 + <i>xylR</i>] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without <i>xylR</i>.</li.<br />
<li>A new PCR of <i>xylR</i> is made, in order to increase the stock.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="21"><br />
<date>Tuesday, August 21st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector <i>E. coli</i> - <i>B. subtilis</i>). Different ligations are made with different proportions of insert and vector. Transformation in NM522 strain.</li><br />
<li>Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.</li><br />
<li>Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li><br />
<li>Results of the second transformation of NM522 by pSB1T3 and RBS-<i>abrB</i> : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Two standard ligations are done :</li><br />
<ul> <br />
<li>pBK7-<i>xylR</i> (<i>xylR</i> cut in X and P sites);</li><br />
<li>pBK24-<i>xylR</i> (<i>xylR</i> cut in E and S sites).</li><br />
</ul><br />
<li>Transformation of pBK7-<i>xylR</i> into NM522 strain.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Wednesday, August 22nd 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 with [Constitutive Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.</li.<br />
<li>New trial of cloning : Digestion, Ligation, transformation to construct :</li><br />
<ul><br />
<li>[Lysostaphin + Dispersin] in pSB1C3;</li><br />
<li>[Lysostaphin + Dispersin] in the shuttle vector (vector for <i>E. coli</i> and <i>B. subtilis,</i>).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 P<sub>xyl</sub> clones were tested, but none had integrated the plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Verification of 6 [<i>xylR</i>-pSB1K3] clones : they only have the vector.</li><br />
<li>Transformation of [pBK24-<i>xylR</i>] into NM522 strain.</li><br />
<li>Results of [pBK7-<i>xylR</i>] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li><br />
<li>Liquid culture of <i>B. subtilis</i> <i>abrB</i> in 5mL of LB broth.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Thursday, August 23rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformations results : All controls are okay.</li><br />
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li><br />
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li><br />
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)</li><br />
<ul><br />
<li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li><br />
<li>The second clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2);</li><br />
<li>The third clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of [P<sub>xyl</sub>+pSB1T3] in NM522.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of [pBK24-<i>xylR</i>] transformation : most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.</li><br />
<li>Extraction of pBK7-<i>xylR</i> from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> <i>abrB</i> in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate').<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Friday, August 24th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A ligation of pBK4 (pUC57 containing the <i>lacI</i> gene) and pBK29 (pSB1A3 containing the <i>sfp</i> and <i>abrB</i> genes) in pBK24 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>After the bad results with <i>xylR</i>, we decided to cut pBK10 plasmid in <i>Sma</i>I site, and ligate with <i>xylR</i>, doing a blunt ligation. We also decided to ligate <i>xylR</i> with <i>xylR</i>, creating a big polymer, which will be like a “pre-ligation” molecule.</li><br />
<li>Extraction of pBK24-<i>xylR</i> from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! Meaning that it’s the first time we manage to ligate <i>xylR</i> successfully.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> <i>abrB</i> strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms.<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date>Saturday, August 25th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of <i>Spe</i>I enzyme, so we could not digest the extracted plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
6 white clones from the ligation pBK24-<i>xylR</i> are purified and put in liquid cultures.<br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date>Sunday, August 26th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Extraction from the 6 white clones (containing <i>xylR</i>-pBK26). Gel electrophoresis is run, but it didn’t show any good clone. <br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date>Monday, August 27th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li><br />
<li>Midiprep : Extraction of P<sub>veg</sub>-dispersin-pSB1C3 of clone 22.</li><br />
<li>BK33 strain was put in storage.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li><br />
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P<sub>xyl</sub>, RBS and <i>sfp</i> gene), <i>abrB</i> gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
No transformation was done, because we ran out of LB broth. <br />
</description><br />
</jour><br />
<br />
<jour nb="28"><br />
<date>Tuesday, August 28th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Meeting at 1:30 pm.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li><br />
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria → Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br /><br />
We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid cultures and then streaked on LB+Cm plates and LB+Amp plates.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Transformation of <i>xylR</i>-pBK10 (blunt end ligation) in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="29"><br />
<date>Wednesday, August 29th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li><br />
<li>Results of the second transformation of Bs 168 strain with pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).</li><br />
<li>We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.</li><br />
<li>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> genes. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.</li><br />
<li>Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using two ligated genes (<i>sfp</i> and <i>abrB</i>) coming from clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classic protocol because we were running out of time and we were behind the schedule.</li><br />
<li>In parallel, we decided to do a standard ligation in order to transfer the <i>lacI</i> gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Transformation of <i>xylR</i>-pBK24 in NM522. The transformation of <i>xylR</i>-pBK24 is done in order to see if <i>xylR</i> was successfully ligated or not. If the bacteria are red, then <i>xylR</i> was not ligated.</li><br />
<li>24 clones containing pBK10-<i>xylR</i> are patched in LB+Amp growth medium, and then incubated.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date>Thursday, August 30th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li><br />
<li>12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.</li><br />
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li><br />
<li>Standard ligation between the shuttle vector pBK26 (pHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.</li><br />
<li>The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.</li><br />
<li>The miniprep confirmed that the ligation containing <i>lacI</i> was successful.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing <i>xylR</i>-pBK10 ligation. The clone is put in liquid culture overnight.</li><br />
<li>Another test is run to confirm that pBK34 plasmid (<i>xylR</i>-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since <i>xylR</i> contains a <i>Nde</i>I restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains <i>xylR</i>. Result : pBK34 doesn’t contain xylem.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date>Friday, August 31st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by <i>Spe</i>I, so the ligation was successful. The four plasmids are further tested for the other 3 iGEM sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digestion was done (<i>Eco</i>RI and <i>Spe</i>I). There was no fragment at 1,000 bp, so the ligation was successful.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li><br />
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li><br />
<li>Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :</li><br />
<ul> <br />
<li>The negative control is ok;</li><br />
<li>The positive control is full of colonies;</li><br />
<li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA. </li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the saturated cultures with transformed bacteria containing the ligation of the <i>sfp</i> and <i>abrB</i> genes. The electrophoresis confirmed that this time the bacteria contained the right plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.</li><br />
<li>The plasmid is digested by <i>Eco</i>RI, <i>Xba</i>I, <i>Spe</i>I and <i>Pst</i>I to find out if the problem comes from a bad site sequence. Result : the <i>Eco</i>RI restriction site is not good, therefore is not recognized by <i>Eco</i>RI enzyme.</li><br />
</ul><br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="September" size="30"><br />
<jour nb="1"><br />
<date>Saturday, September 1st 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the manufacturer protocol and another trial is made with higher concentration.</li><br />
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li><br />
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li><br />
<li>Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into <i>E. coli</i> NM522 strain.</li><br />
<li>6 other clones with the transformed 3A ligation (done on August 29th) are screened.</li><br />
<li>Standard ligation of the <i>abrB</i> gene and the plasmid containing <i>lacI</i> in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="2"><br />
<date>Sunday, September 2nd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated overnight.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized, followed by transformation in <i>E. coli</i> NM522 strain.</li><br />
<li>Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.</li><br />
<li>12 clones for each ligation (done the day before) are screened.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date>Monday, September 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Midiprep of the cloned shuttle vectors (containing the iGEM linker).</li><br />
<li>Miniprep of pBK26 shuttle vector is done in order to increase the stock.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>No clones are obtained on the plate lysostaphin+dispersin when we followed the manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.</li><br />
<li>Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.</li><br />
<li>A transformation is made with our cells of their positive control.</li><br />
<li>Transformations of Bs 168 :<br />
<ul><br />
<li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li><br />
<li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li><br />
</ul><br />
The bacteria are spread on LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).<br /><br />
The transformation of NM522 with ligation Dispersin in pBK26 was successful.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li><br />
<li>6 clones transformed with the mixture of two plasmids are screened.</li><br />
<li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the <i>lacI</i> and <i>abrB</i> genes). The clone is put in storage under the reference pBK39.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date>Tuesday, September 4th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 1.3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.</li><br />
<li>Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.</li><br />
<li>Results of the transformations of Bs 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li><br />
<li>Standard ligations between :<br />
<ul><br />
<li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Pst</i>I;</li><br />
<li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Eco</i>RI.</li><br />
</ul><br />
These two ligation products are transformed in NM522 strain.</li><br />
<li>Acrylamide gels and samples are prepared for SDS-PAGE.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis showed that 5 out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with <i>Eco</i>RV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A PCR is run in order to get RBS-<i>xylR</i>. The PCR failed (not enough DNA-polymerase).<br />
</description><br />
<titre>Biofilm</titre><br />
<description><br />
<ul><br />
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li><br />
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li><br />
<li>Same plate as the one above + crystal violet.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date>Wednesday, September 5th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK22 (<i>sfp</i> gene) and pBK39 (<i>abrB</i>-<i>lacI</i>) and transformation into the <i>E. coli</i> NM522 strain.</li><br />
<li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date>Thursday, September 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD<sub>600</sub> of the liquid cultures is. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Clones obtained on lysostaphin-dispersin plates are put in liquid culture.</li><br />
<li>Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD<sub>600</sub>600 measurements in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The Bs 168 strain is transformed by :<br />
<ul><br />
<li>pBK 25 (= modified shuttle vector pHT304);</li><br />
<li>pBK 26 (= modified shuttle vector pHT315);</li><br />
<li>pBK 28 (= Lysostaphin in the modified shuttle vector pHT304);</li><br />
<li>pBK 38 (= Lysostaphin in the modified shuttle vector pHT315);</li><br />
<li>Dispersin in pBK26.</li><br />
</ul><br />
We spread 200 µL of each transformed cells on LB + Erythromycin ([Ery] = 16 µg/mL) plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones transformed with the ligation pBK22 and pBK39 are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
2 new PCR are done to have <i>xylR</i>, but they failed. <br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Friday, September 7th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.</li><br />
<li>Result of the transformation of Bs 168 : </li><br />
<ul><br />
<li>All the negative control plates are clear : the erythromycin concentration is high enough to do a selection;</li><br />
<li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to extract and analyze their DNA;</li><br />
</ul><br />
<li>Transformation of Bs 168 : new trial by electroporation. As the last transformation, the Bs 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li><br />
<li>A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (<i>sfp</i>, <i>abrB</i> and <i>lacI</i>) would be higher.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A new RBS-<i>xylR</i> PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Saturday, September 8th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Miniprep of the clones Bs 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for <i>E.coli</i> with addition of Lysozyme to the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel → Maybe the DNA isn’t concentrated enough ?<br /><br />
New clones are put in liquid cultures in order to make other minipreps. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of P<sub>lac</sub>-RBS-<i>xylR</i> and then transformation in NM522.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Each of the 24 wells of two microtiter plates are inoculated with 2mL of a suspension obtained from diluting the liquid culture 100 times with TSB enriched with 1% glucose The plate is incubated during 24 hours at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Sunday, September 9th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones Bs 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li><br />
<ul><br />
<li>Bs 168 clone transformed by pBK25 has the right plasmid !! =)</li><br />
<li>Bs 168 clone transformed by pBK28 has the right plasmid !! <strong>BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</strong></li><br />
<li>Bs 168 clones transformed by pBK26 and dispersin in pBK26 : nothing on the gel... </li><br />
</ul><br />
<li>Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + Ery plates, with two different concentrations : [Ery]=10 µg/mL and [Ery]=15 µg/mL.</li><br />
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li><br />
<li>Thanks to the good results, we put in storage the transformed <i>Bacillus</i> under the reference :</li><br />
<ul><br />
<li>BK41 : Bs 168 + pBK25</li><br />
<li>BK42 : Bs 168 + pBK28</li><br />
<li>BK43 : Bs 168 + pBK26 (to confirm)</li><br />
<li>BK44 : Bs 168 + Dispersin in pBK26 (to confirm)</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The supernatant from the two microtiter plates are decanted. The wells of a third microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the dispersin. The supernatants are diluted in order to test different concentrations of the supernatants. The wells of a fourth microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the lysostaphin.<br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Monday, September 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.</li><br />
<li>Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the <i>E. coli</i> NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li><i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.</li><br />
<li>According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD<sub>600</sub> of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD<sub>600</sub>.</li><br />
<li>New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.</li><br />
</ul><br />
</description><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
<ul><br />
<li>Measurement of the NM522 + P<sub>lac</sub> kinetics (with Cm) during 12h in a 96 well plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)</li><br />
<li>In the same 96 well plate NM522 + P<sub>xyl</sub> kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)</li><br />
<li>Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plates prepared the day before are analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can see that the biofilm is not adherent enough and it is destroyed no matter what kind of supernatant is tested. Unfortunately, we cannot say what the effect of the dispersin or lysostaphin is using this kind of test.<br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Tuesday, September 11th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm (plate 2).</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).</li><br />
<li>Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones per transformation are screened.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. <i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.</li><br />
<li>SDS-PAGE is done using pellets, to find a good concentration.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Wednesday, September 12th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation. Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.</li><br />
<li>Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately, the screened clones did not integrate the expected plasmid.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture with the new protocol. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. There is a problem with the results : the control is not good.</li><br />
<li>Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>5 test tubes containing a lamella are inoculated with the culture prepared the day (<i>S. epidermidis</i>) before diluted 1:100 with TSB supplemented with 1% glucose.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Thursday, September 13th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding of 2 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>OD<sub>600</sub> measurements with filtered supernatants.</li><br />
<li>Mobility test of Bs168 on agar 0.25% plate.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.</li><br />
<li>Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Another 12 clones per transformation were screened (from the transformation made on 10th September).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in tanks to test dispersin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce dispersin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. Dispersin has no effect on <i>S. epidermidis</i> culture according to the results.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>One of the tubes and lamella prepared the day before containing <i>S. epidermidis</i> cultures is quantified using crystal violet staining and OD<sub>600</sub> is measured. <i>S. epidermidis</i> cells are quite adherent on glass surfaces.</li><br />
<li>The supernatant of the four other tubes are decanted and tubes are filled with the supernatants produced by the four liquid cultures of <i>B. subtilis</i> prepared the day before in order to test the effect of lysostaphin and dispersin.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Friday, September 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).<br /><br />
Moreover, OD<sub>600</sub> of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.</li><br />
<li>TG1 (<i>E. coli</i>) strain containing pJIM2241 shuttle vector is put in storage (BK47).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600s</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. The strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li>SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The four tubes and lamella prepared the day before are quantified using crystal violet staining and OD<sub>600</sub>is measured. The same result is obtained : <i>S. epidermidis</i> biofilms are not adherent enough and biofilms are destroyed when the growth medium is changed.<br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Saturday, September 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the 2 shuttle vectors pBK35 and pBK36.<br />
</description><br />
<br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in 96 well plate to test impact of lysostaphin and dispersin on a culture of <i>S. epidermidis</i> (test during 12 hours).<br />
</description><br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
LB plates containing a 0.3% agarose gel are inoculated with 5 μL of a 5 hour liquid culture of <i>B. subtilis</i> containing the plasmids used in transformations.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligations between the plasmid pBK42 containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] and the shuttle vectors pBK35 and pBK36 into the NM522 strain .<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Sunday, September 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Screening of 12 clones per <i>Bacillus</i> transformation.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 15 clones transformed with the ligation pBK42+pBK35 and pBK42+pBK36<br />
</description><br />
<br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
The diameter of the disks formed by <i>B. subtilis</i> is measured. The fact of introducing the plasmids did not impact the swarming mobility of our bacteria.<br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Monday, September 17th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Erythromycin at different concentrations was spread on LB plates with in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="18"><br />
<date>Tuesday, September 18th 2012</date><br />
titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened.<br />
</description><br />
<br />
<br />
</jour><br />
<br />
<jour nb="19"><br />
<date>Wednesday, September 19th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.<br />
</description><br />
<br />
<br />
<br />
<br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Thursday, September 20th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector. <br />
</description><br />
<br />
<br />
</jour><br />
<br />
<br />
<br />
<jour nb="21"><br />
<date>Friday, September 21st 2012</date><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
A 7-hour-long kinetics is made with P<sub>lac</sub> and P<sub>xyl</sub> to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used.<br />
P<sub>lac</sub> = IPTG 0.5% and P<sub>xyl</sub> = xylose 0.5%.<br /><br />
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
SDS-PAGE is done in order to detect Dispersin in <i>B. subtilis</i> and <i>E. coli</i>'s supernatants. The gels were not conclusive, and were not good due to an ammonium persulfate problem.<br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Saturday, September 22th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
12 clones transformed with the shuttle vectors are screened.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A new SDS-PAGE is done for dispersin. A migration problem persists, due to ammonium persulfate. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
24 clones transformed with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened.<br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Sunday, September 23th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing 2 mL liquid LB medium are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A last SDS-PAGE is run. This time the problems were successfully solved. But the gels were not conclusive due to a low dispersin concentration.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Monday, September 24th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Both strains grew in all tubes. However, there is a difference concerning OD<sub>600</sub> between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1.5 mg/mL) in order to make sure there is a notable difference between the two strains. </li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<li> In order to characterize the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] two tests are made. To confirm the presence of <i>sfp</i> gene, we made emulsions with the filtered supernatant of the transformed strains and sunflower oil. Concerning <i>abrB</i> gene, a biofilm formation test was made in a 24-well microplate in order to compare the transformed strain to the wild-type strain. <br />
</li><br />
</description><br />
</jour><br />
<jour nb="25"><br />
<date>Tuesday, September 25th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes; <br />
</description><br />
</jour><br />
<br />
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<h1>The Notebook</h1><br />
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<h1>Our collections</h1><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/notebookTeam:Lyon-INSA/notebook2012-10-25T19:51:51Z<p>Suxiaohui: </p>
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$(".textContent").append("<div class='descExp' id='descExp"+k+"'></div>");<br />
$("#descExp"+k).append(jour[day].getElementsByTagName("description")[k].innerHTML); <br />
} <br />
globalDay = day;<br />
}<br />
if (!foundDay) {<br />
<br />
var monthIndexToTest = 0;<br />
var actionType;<br />
if (day - globalDay > 0) {<br />
//day bigger so going to next month<br />
monthIndexToTest = i + 1;<br />
actionType = "asc";<br />
} <br />
else {<br />
//day smaller so going to previous month<br />
monthIndexToTest = i - 1;<br />
actionType = "desc";<br />
}<br />
<br />
if (monthIndexToTest<x.length)<br />
{ <br />
if (x[monthIndexToTest].getElementsByTagName("jour").length>0) {<br />
selectMonth(x[monthIndexToTest].getAttribute("name"),actionType);<br />
} <br />
else<br />
{<br />
globalDay=x[i].getElementsByTagName("jour").length; <br />
} <br />
}<br />
else<br />
{<br />
globalDay=x[i].getElementsByTagName("jour").length; <br />
}<br />
}<br />
} <br />
}<br />
<br />
}<br />
<br />
<br />
function selectMonth(month_name,type) <br />
{<br />
<br />
$(".month").removeClass("BNSelected");<br />
$("#"+month_name).addClass("BNSelected");<br />
<br />
$(".textContent").text("");<br />
<br />
var xmlDoc=document.getElementById("xmldata")<br />
var day=0;<br />
var x=xmlDoc.getElementsByTagName("month");<br />
var monthIndex = -1;<br />
for (i=0;i<x.length;i++)<br />
{<br />
if (x[i].getAttribute("name")==month_name)<br />
{ <br />
if (type=="desc")<br />
{<br />
day=x[i].getElementsByTagName("jour").length-1;<br />
}<br />
var jour=x[i].getElementsByTagName("jour");<br />
$(".textContent").append("<div class='dateExp'></div>");<br />
$(".dateExp").append(jour[day].getElementsByTagName("date")[0].childNodes[0].nodeValue); <br />
<br />
for (j=0;j<jour[day].getElementsByTagName("titre").length;j++)<br />
{<br />
$(".textContent").append("<div class='titreExp' id='titreExp"+j+"'></div>");<br />
$("#titreExp"+j).append(jour[day].getElementsByTagName("titre")[j].childNodes[0].nodeValue); <br />
$(".textContent").append("<div class='descExp' id='descExp"+j+"'></div>");<br />
$("#descExp"+j).append(jour[day].getElementsByTagName("description")[j].innerHTML); <br />
} <br />
<br />
monthIndex=i;<br />
<br />
<br />
} <br />
} <br />
<br />
/*MODIF LUCAS*/<br />
//initialisation de la liste de jour pour le mois selectionné<br />
var dayCounter = 1;<br />
if (monthIndex != -1) { <br />
var monthSize = parseInt(x[monthIndex].getAttribute("size"));<br />
$("#monthDays ul").children().remove();<br />
for (j=0;j<jour.length;j++) {<br />
var xmlDayNumber = jour[j].getAttribute("nb");<br />
if (xmlDayNumber != null){if (dayCounter <= monthSize) {<br />
// nombre seul<br />
while(!(dayCounter == xmlDayNumber || dayCounter == monthSize + 1)){<br />
<br />
$("#monthDays ul").append("<li>"+ dayCounter +"</li>");<br />
dayCounter++;<br />
}<br />
<br />
//nombre avec un lien<br />
$("#monthDays ul").append("<li><a href='#' onClick='selectDayAnimate(\""+ month_name + "\", " + xmlDayNumber + ", true, this);" + "return false;'>" + xmlDayNumber +"</a></li>");<br />
dayCounter++;<br />
}else {<br />
$("#monthDays ul").append("<li>"+ dayCounter +"</li>");<br />
dayCounter++;<br />
}<br />
<br />
} else {<br />
$("#monthDays ul").append("<li>"+ dayCounter +"</li>");<br />
dayCounter++;<br />
}<br />
}<br />
while(dayCounter != (monthSize + 1) ) {<br />
$("#monthDays ul").append("<li>"+ dayCounter +"</li>");<br />
dayCounter++;<br />
}<br />
}<br />
else {<br />
//le mois m'existe pas dans le xml, la liste n'a pas de liens<br />
$("#monthDays ul").children().remove();<br />
while(dayCounter != 32) {<br />
$("#monthDays ul").append("<li>"+ dayCounter +"</li>");<br />
dayCounter++;<br />
} <br />
}<br />
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globalDay=day;<br />
globalMonth=month_name;<br />
}<br />
<br />
function nextDay(month_name,day) <br />
{<br />
$(".textContent").html("");<br />
var xmlDoc=document.getElementById("xmldata")<br />
var newDay=day+1;<br />
var x=xmlDoc.getElementsByTagName("month");<br />
<br />
for (i=0;i<x.length;i++)<br />
{<br />
if (x[i].getAttribute("name")==month_name)<br />
{<br />
var jour=x[i].getElementsByTagName("jour");<br />
if (jour.length>newDay)<br />
{<br />
<br />
$(".textContent").append("<div class='dateExp'></div>");<br />
$(".dateExp").append(jour[newDay].getElementsByTagName("date")[0].childNodes[0].nodeValue); <br />
<br />
for (j=0;j<jour[newDay].getElementsByTagName("titre").length;j++)<br />
{<br />
$(".textContent").append("<div class='titreExp' id='titreExp"+j+"'></div>");<br />
$("#titreExp"+j).append(jour[newDay].getElementsByTagName("titre")[j].childNodes[0].nodeValue); <br />
$(".textContent").append("<div class='descExp' id='descExp"+j+"'></div>");<br />
$("#descExp"+j).append(jour[newDay].getElementsByTagName("description")[j].innerHTML); <br />
} <br />
<br />
<br />
<br />
globalDay=newDay; <br />
<br />
<br />
}<br />
else if (i+1<x.length)<br />
{ <br />
if (x[i+1].getElementsByTagName("jour").length>0)<br />
{selectMonth(x[i+1].getAttribute("name"),"asc");} <br />
else<br />
{<br />
globalDay=x[i].getElementsByTagName("jour").length; <br />
} <br />
}<br />
else<br />
{<br />
globalDay=x[i].getElementsByTagName("jour").length; <br />
}<br />
} <br />
}<br />
<br />
<br />
<br />
}<br />
<br />
function previousDay(month_name,day) <br />
{<br />
<br />
$(".textContent").html("");<br />
var xmlDoc=document.getElementById("xmldata")<br />
var newDay=day-1;<br />
var x=xmlDoc.getElementsByTagName("month");<br />
for (i=0;i<x.length;i++)<br />
{<br />
if (x[i].getAttribute("name")==month_name)<br />
{<br />
if (day>0)<br />
{<br />
var jour=x[i].getElementsByTagName("jour");<br />
<br />
$(".textContent").append("<div class='dateExp'></div>");<br />
$(".dateExp").append(jour[newDay].getElementsByTagName("date")[0].childNodes[0].nodeValue); <br />
for (j=0;j<jour[newDay].getElementsByTagName("titre").length;j++)<br />
{<br />
$(".textContent").append("<div class='titreExp' id='titreExp"+j+"'></div>");<br />
$("#titreExp"+j).append(jour[newDay].getElementsByTagName("titre")[j].childNodes[0].nodeValue); <br />
$(".textContent").append("<div class='descExp' id='descExp"+j+"'></div>");<br />
$("#descExp"+j).append(jour[newDay].getElementsByTagName("description")[j].innerHTML); <br />
} <br />
<br />
<br />
<br />
globalDay=newDay;<br />
}<br />
else if (i>0)<br />
{ <br />
if (x[i-1].getElementsByTagName("jour").length>0)<br />
{selectMonth(x[i-1].getAttribute("name"),"desc");} <br />
}<br />
else <br />
{<br />
globalDay=-1;<br />
}<br />
} <br />
} <br />
<br />
<br />
}<br />
<br />
<br />
</script><br />
<br />
<head><br />
</head><br />
<br />
<body><br />
<br />
<div id="xml" style="display:none;"><br />
<xml id='xmldata' style='display:none;'><br />
<?xml version="1.0" encoding="ISO-8859-1"?><br />
<!-- Edited by XMLSpy --><br />
<project><br />
<month name="July" size="31"><br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 2nd 2012</date><br />
<description>Liquid culture (5 mL LB medium) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description> <br />
</jour><br />
<jour nb="3"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 3rd 2012</date><br />
<description><p>Dilution of 100 µL saturated culture in 5 mL LB medium. </p><br /><br />
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 0.3). </p><br /><br />
Transformation of the NM522 strain (this experiment was repeated 3 times). <br />
<ul><br />
<li>For the positive control the pSB1C3 plasmid was used;</li><br />
<li>For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).</li><br />
</ul><br />
The transformed bacteria were selected on chloramphenicol (Cm) plates.<br />
</description><br />
</jour><br />
<jour nb="4"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 4th 2012</date><br />
<description><br />
Transformation analysis :<br/><br/><br />
<ul><br />
<li>Positive control : lots of colonies;</li><br />
<br />
<li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the two other negative control plates;</li><br />
<br />
<li>Test plate : between 1 and 8 were observed.</li></ul><br />
<br/><br />
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/><br />
<br />
The antibiotic resistance to Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin of the following strains was tested : BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS <i>abrB</i>.<br />
</description><br />
</jour> <br />
<jour nb="5"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 5th 2012</date><br />
<description><br />
Antibiotics resistance tests :<br/><br/><br />
<ul><br />
<li>Bs 168 : no resistance;</li><br />
<li>Bs 168 M cherry : no resistance;</li><br />
<li>Bs 168 GFP : no resistance;</li><br />
<li>Bs <i>abrB</i> : Cm resistant;</li><br />
<li>Bs 168 lysostaphin PWG100 : no resistance;</li><br />
<li><i>S. epidermidis</i> : Tet resistant.</li><br />
</ul><br />
<br/><br />
Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after <i>Eco</i>RI digestion)<br />
<br />
</description><br />
</jour><br />
<jour nb="6"><br />
<titre>For all purposes</titre><br />
<date> Friday, July 6th 2012</date><br />
<description><br />
<br />
Phone conference with Romain Briandet.<br/><br/><br />
<br />
Terminator was retrieved from plate 1 well 13D.<br/><br/><br />
Long meeting.<br />
<br />
</description><br />
</jour><br />
<jour nb="9"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 9th 2012</date><br />
<description><br />
<ul><br />
<li>pBBa_I742123 was put in storage (under the reference pBK1).</li><br />
<li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB medium + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li><br />
<li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li><br />
<li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li><br />
<li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</li><br />
<li>Liquid cultures of the following strains were made : Bs 168 in LB, Bs <i>abrB</i> in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul><br />
<br />
</description><br />
</jour><br />
<jour nb="10"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 10th 2012</date><br />
<description><br />
<ul><br />
<li> The following parts were put in storage :</li><br />
<ul><br />
<li>Lysostaphin in pUC57 Amp resistant (pBK2);</li><br />
<li>Dispersin in pUC57 Amp resistant (pBK3);</li><br />
<li>Surfactin part 2 (RBS-<i>lacI</i>-terminator) in pUC57 Amp resistant (pBK4).</li><br />
</ul><br />
<li> and the following strains (in LB broth supplemented with required antibiotic) :</li><br />
<ul> <br />
<li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li><br />
<li>Bs <i>abrB</i> = BK2 (Cm<sup>R</sup>);</li><br />
<li>Bs 168 = BK3.</li><br />
</ul><br />
<li>Ligation of Dispersin and Lysostaphin biobricks :</li><br />
<ul><br />
<li>digestion of pBK2 with the restriction enzymes <i>Eco</i>RI and <i>SpeI</i>;</li><br />
<li>digestion of pBK3 with the restriction enzymes <i>PstI</i> and <i>XbaI</i>;</li><br />
<li>digestion of pBK4 with the restriction enzymes <i>EcoRI</i> and <i>PstI</i>;</li><br />
<li>3A ligation of the digested parts;</li><br />
<li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2.1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.</li></ul><br />
<li>Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 bands are observed : <strong>PROBLEM. Digestion is made again with E, P and E+P → There are 2 <i>PstI</i> sites !!! WRONG PLASMID</strong></li><br />
<br />
<li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li><br />
<li>Design and order of the primers for the constitutive promoter (part BBa_K143012).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 11th 2012</date><br />
<description><br />
<ul><br />
<li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain.</li><br />
<li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain. <i>(NB : the clones were pink which means that the part contains a RFP gene)</i></li><br />
<li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain.</li><br />
<li>Extraction of pUC57-Lysostaphin/pUC57-Dispersin/pUC57-<i>lacI</i> from transformed NM522 strains.</li><br />
<li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the color of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 12th 2012</date><br />
<description><br />
<ul><br />
<li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li><br />
<li>Reception and storage of the primers (for the amplification of the BBa_K143012 part).</li><br />
<li>The transformed NM522 strain with the part BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit.</li><br />
<li>Extractions with a miniprep kit are made :<br />
<ul><br />
<li>4 clones containing the P<sub>lac</sub> promoter (1,2,3,4);</li><br />
<li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li><br />
<li>3 clones containing theTerminator (1,2,3);</li><br />
<li>4 clones containing the gene for Dispersin;</li><br />
<li>4 clones containing the gene for Lysostaphin;</li><br />
<li>4 clones containing the gene for <i>lac</i>I.</li><br />
</ul><br />
Then a digestion is made on a 0.7% agarose gel.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<titre>Kill</titre><br />
<date> Friday, July 13th 2012</date><br />
<description><br />
<ul><br />
<li>PCR of the constitutive promoter (part BBa_K143012) → the PCR did not work so we ran a new PCR with a more diluted promoter solution.</li><br />
<li>The following strains are put in storage:<br />
<ul><br />
<li>NM522 containing <i>lacI</i>-pUC57;</li><br />
<li>NM522 containing RBS-pUC57;</li><br />
<li>NM522 containing dispersin-pUC57;</li><br />
</ul><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date> Monday, July 16th 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
<ul><br />
<li>PCR of constitutive promoter → it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li><br />
<li>Purification of the PCR product.</li><br />
<li>Gel electrophoresis of lysostaphin.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation of B0015 terminator.</li><br />
<li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date> Tuesday, July 17th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Long morning meeting to discuss results and to write some posters in a frenzied psychopath way in order to remember our tasks.</li><br />
<li>Ligation of promoter-RBS-<i>gfp</i> in plasmid.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168: buffers preparation.</li><br />
<li>Failure of B0015 transformation.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/<i>gfp</i> (of <i>E. coli</i>) using a 3A ligation followed by transformation of the ligation in the NM522 strain;</li><br />
<li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> in 5mL of TSB (conditions described in protocol “Tests on Staphylococcus aureus biofilms in 24 well plate” ).</li><br />
<li>A bacterial suspension of <i>S. epidermidis</i> with OD<sub>600</sub> close to 0.132 is prepared from a Petri dish containing <i>S. epidermidis</i>. A microtiter plate is inoculated with 2 mL of the suspension diluted 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="18"><br />
<date> Wednesday, July 18th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid → extraction failure.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168 (Kit Genomic DNA from tissue).</li><br />
<li>Transformation of pBK5 in <i>B. subtilis</i> <i>abrB</i> failed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation results of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;</li><br />
<li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis : there is still a smear. However, the bands are as expected.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i>.</li><br />
<li>A microtiter plate is inoculated with 2mL of a suspension obtained from diluting the liquid culture 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="19"><br />
<date> Thursday, July 19th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work.</li><br />
<li>A 50µg/mL erythromycin solution is made in order to test the strains’ resistance;</li><br />
<li>Transformation of <i>yfp</i>, <i>gfp</i> and <i>cfp</i> (from iGEM plates) in NM522;</li><br />
<li>B0015 transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth supplemented with Tetracyclin.</li><br />
<li>The fluorescence test of the transformed bacteria containing [dispersin+RBS/<i>gfp</i>+pSB1T3] confirm that the promoter is functional.</li><br />
<li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i> and that the fact of cultivating the strain in broth or on solid medium has no impact on the biofilm's quality.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date> Friday, July 20th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and promoter+pSB1C3 OK → 6 clones are isolated on a LB+Cm plate.</li><br />
<li>Quantification and gel electrophoresis of <i>B. subtilis</i> genomic DNA.</li><br />
<li>Antibiotic testing of <i>B. thuringensis</i> 407 <i>gfp</i> strain and other strains in LB+Ery growth medium.</li><br />
<li>Failure of transforming pBK5 in <i>B. subtilis</i> 168 with the same protocol as previously. New task : find a new protocol.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin.</li><br />
<li>Ligation Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.</li><br />
<li>Ligation Lysostaphin in pSB1C3 and transformation.</li><br />
<li>Isolation of 6 clones of the Terminator transformation.</li><br />
<li>Transformation results of fluorescent genes : ok, except <i>yfp</i>. </li><br />
<li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date> Monday, July 23rd 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Selected clones (NM522+pBK6) are red, meaning that P<sub>veg</sub> promoter and RBS from <i>B. subtilis</i> are recognized by <i>E. coli</i>’s ribosome. 4 clones are streaked in LB+Cm.</li><br />
<li>LB+Ery and TSB+Tet Petri plates are made.</li><br />
<li><i>yfp</i> transformation was successful.</li><br />
<li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li><br />
<li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li><br />
<li>Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results</li><br />
<ul><br />
<li>Promoter+Dispersin in pIG23 : nothing on the plate;</li><br />
<li>Lysostaphin, negative control contains bacteria so the plate is put in junk.</li><br />
</ul><br />
<li>New digestions, ligations,transformations:</li><br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57;</li><br />
<li>Dispersin in pSB1C3;</li><br />
<li>Lysostaphin in pSB1C3;</li><br />
<li>Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).</li><br />
</ul><br />
<li>Streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promoter and the RBS specific to the <i>Bacillus subtilis</i> strain are recognized by the RNA polymerase of <i>E. coli</i>.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR of xylR. Gel electrophoresis : the PCR didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date> Tuesday, July 24th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.</li><br />
<li>Extraction of <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ; test → OK : put in storage.</li><br />
<li>Extraction of the pHT315 GFP plasmid of <i>B. thuringiensis</i> 407 GFP to build a shuttle vector <i>B. subtilis</i> ↔ <i>E. coli</i>. Transformation in NM522 strain and selection on LB+Ampicillin medium : ok.</li><br />
<li><i>S. epidermidis</i> is put in storage in TSB medium under the reference BK15.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results:<br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;</li><br />
<li>Promoter+Dispersin : nothing on the plate;</li><br />
<li>Dispersin in pSB1C3 : a lot of clones;</li><br />
<li>Lysostaphin in pSB1C3 : a lot of clones.</li><br />
</ul><br />
Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (because there are too many clones!)</li><br />
<br />
<li>Extraction of the plasmidic DNA [Promoter in pSB1C3].</li><br />
<li>Extraction of pBK6 from the transformed strain.</li><br />
<li>Extraction of the [Promoter+RBS+<i>gfp</i>] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by <i>Eco</i>RI and <i>Pst</i>I : gel ok. Congrats (pBK12) → 122,6 ng/µL.</li><br />
<li>Extraction of the pBK6 plasmid (P<sub>veg</sub>+RBS+RFP in pSB1C3). The electrophoresis confirms the plasmid’s structure.</li><br />
<li>Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New PCR of xylR with some modifications in the protocol : the PCR didn’t work.<br />
<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date> Wednesday, July 25th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Strains are put in storage :</li><br />
<ul><br />
<li><i>S. epidermidis</i> in TSB+Tet (BK17);</li><br />
<li><i>B. thuringensis</i> 407 GFP in LB+Ery (BK16).</li><br />
</ul><br />
<li>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).</li><br />
<li>Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (GFP or not).</li><br />
<li>The plasmid extraction protocol from <i>B. subtilis</i> was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of the extracted plasmidic DNA of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.</li><br />
<li>Selection of 4 clones transformed by [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.</li><br />
<li>Miniprep to extract [Lysostaphin+Cm] and [Dispersin+Cm] → Digestion.</li><br />
<li>3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).</li><br />
<li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on LB+Tet plates having a biofilm already established and in development of <i>S. epidermidis.</i></li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR on the <i>B. subtilis</i> 168 genome using universal primers of Phil Oger, and positive control with <i>Bulkhoderia cepacia</i> genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of <i>B. subtilis</i> 168. <br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date> Thursday, July 26th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for <i>B. subtilis</i> and <i>E. coli</i>).</li><br />
<li>gDNA extraction with 2 different protocols : the first for <i>Bacillus subtilis</i> 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.</li><br />
<li>PCR of rRNA 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.</li><br />
<li>NM522 + <i>gfp</i>, <i>cfp</i> and <i>yfp</i> in storage.</li><br />
<li>pHT315 GFP put in storage under the reference pBK18.</li><br />
<li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other verification by extracting their plasmidic DNA (but they are not good again).</li><br />
<li>Extraction of the plasmidic DNA of the clones transformed with Lysostaphin+Dispersin in pBK2 and verification by electrophoresis gel. The clones are not right.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A assembly RBS-<i>gfp</i> (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date> Friday, July 27th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
Extraction of transformed clones (NM522/pHT304 and NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites <i>Xba</i>I, <i>Eco</i>RI and <i>Pst</i>I as expected. However, there is a <i>Spe</i>I site which had not been mapped.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.</li><br />
<li>Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.</li><br />
<li>New 3A ligation with Lysostaphin/Terminator/pSB1C3 and transformation in NM522 strain.</li><br />
<li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 and pBK17.</li><br />
<li>Failure of transformation of L1 in NM522.</li><br />
<li>3A assembly of RBS-<i>cfp</i> (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li><br />
<li>3A assembly of <i>sfp</i>-part2(<i>lacI</i>)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>PCR of <i>xylR</i> from gDNA extracted yesterday. Test on gel : successful PCR !!</li><br />
<li>3A ligation of RBS (pBK7) and <i>xylR</i> (produced by PCR) in pSB1C3 ( ! the vector plasmid has the same resistance as the plasmid containing the RBS...).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date> Monday, July 30th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li><strong>It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire…).</strong></li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion test and gel of pBK2 (promoter + lysostaphin) by <i>Eco</i>RI and <i>Spe</i>I, pBK3 (dispersin + terminator) by <i>Xba</i>I and <i>Pst</i>I and pSB1C3 by <i>Eco</i>RI and <i>Pst</i>I.</li><br />
<li>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel → the 3 clones aren’t right.</li><br />
<li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD<sub>600</sub> = 0.5, dilution the culture x100, 1000 and 10 000).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of L1, L2 and IV in NM522.</li><br />
<li>Transformation of <i>sfp</i> and <i>abrB</i> in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Purification of <i>xylR</i>, produced by PCR.<br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date> Tuesday, July 31st 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin).<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i><i>sfp</i></i> parts.</li><br />
<li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of RBS and <i>xylR</i> and transformation in NM522 strain.<br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="August" size="31"><br />
<jour nb="1"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, August 1st 2012</date><br />
<description><br />
<br />
<p>The transformation protocol for <i>Bacillus</i> was tested using the pHT315 GFP plasmid extracted from the <i>B. thuringiensis</i> BT407GFP strain.</p><br />
<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>Lysostaphin+terminator : nothing;</li><br />
<li>Lysostaphin+dispersin : a lot of clones.</li><br />
</ul><br />
Cultures in LB broth of Lysostaphin+Dispersin are launched.</li><br />
<li>Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.</li><br />
<li>pUC57 with Dispersin put in storage : pBK3.</li><br />
<li>Lysostaphin test returns ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The strain NM522/pBK6 was put in storage under the reference BK22.</li><br />
<li>Transformations of the NM522 strain with the ordered constructions (<i>sfp</i> and <i>abrB</i> in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li><br />
<li>Quantification of <i>sfp</i> and <i>abrB</i> constructions provided by Genecust using the Nanodrop.</li><br />
<li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li><br />
</ul><br />
</description> <br />
</jour><br />
<br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Thursday, August 2nd 2012</date><br />
<description><br />
<ul><br />
<li>The transformation of the <i>Bacillus</i> strain failed because of contaminated LB broth, so a new transformation was attempted.</li><br />
<li>pHT304 and pHT315 plasmids supernumerary site <i>Spe</i>I deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dispersin clones digestion : clones not okay.</li><br />
<li>Ligations of :<br />
<ul><br />
<li>Constitutive promoter in Cm iGEM plasmid;</li><br />
<li>Dispersin in Cm iGEM plasmid.</li><br />
</ul><br />
Ligation were verified by electrophoresis.</li><br />
<li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the <i>Bacillus</i> colonies where <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li><br />
<li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the <i>abrB</i> construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li><br />
<li>New ligation of RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <I>sfp</i> and <i>abrB</i> in NM522 strain didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date> Friday, August 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Purification of the NM522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li><br />
<li>The results of <i>Bacillus</i> transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li><br />
<li>A lot of clones for the transformation of the pHT304 and pHT315 plasmids (without <i>Spe</i>I site) on LB+Amp plates → Purification of 12 clones each.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li><br />
<li>Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).</li><br />
<li>BK12 strain verification : spreading on LB + Amp plate.</li><br />
<li>Ligation of Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li><br />
<li>Ligation were verified by electrophoresis.</li><br />
<li>Transformation of Promoter+Dispersin ligation.</li><br />
<li>PCR to check the presence of the promoter in the Promoter in Cm iGEM plasmid clones.</li><br />
<li>Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill <i>Bacillus</i>, 500 µL of Bs 168 pWG100 supernatant, 500 µL of <i>S. epidermidis</i> with OD<sub>600</sub>=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD<sub>600</sub> with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+<i>cfp</i>+pSB1K3).</li><br />
<li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+<i>gfp</i>+pSB1K3).</li><br />
<li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li><br />
<li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New ligation between RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date> Saturday, August 4th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>The negative control is ok;</li><br />
<li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li><br />
</ul><br />
8 clones for each transformation are chosen and spread on LB+Cm and LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformations of <i>abrB</i> and <i>sfp</i> are a success !! :D</li><br />
<li>Liquid cultures of <i>abrB</i> and <i>sfp</i> clones are launched to do plasmid extractions.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Sorting of the RBS + <i>xylR</i> clones to eliminate the clones containing two relegated plasmids.<br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date> Sunday, August 5th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 S.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Plasmid extractions from 3 clones NM522/<i>abrB</i> and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp).<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Liquid cultures of RBS+<i>xylR</i> clones are launched to do plasmid extractions.<br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date> Monday, August 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Plasmid extraction from 6 clones Bs 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li><br />
<li>Miniprep and digestions by <i>Spe</i>I of the plasmid extracted from the 6 clones of NM522 with pHT304 S and NM522 with pHT315 S : Failure ! The <i>Spe</i>I site is still in the plasmids :’( </li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Selection of clones growing on LB+Cm plates and not on LB+Amp plates :<br />
<ul><br />
<li>4 clones Dispersin in pSB1C3;</li><br />
<li>7 clones Promoter pSB1C3;</li><br />
<li>5 clones Promoter+Dispersin in pSB1C3.</li><br />
</ul><br />
Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li><br />
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on LB+Cm and LB+Amp to test their resistance.</li><br />
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of the following ligations, in NM522 strain :</li><br />
<ul><br />
<li>pBK7+pBK13+pSB1K3 (RBS+<i>cfp</i> in Kanamycin resistant backbone);</li><br />
<li>pBK7+pBK14+pSB1K3 (RBS+<i>gfp</i> in Kanamycin resistant backbone).</li><br />
</ul><br />
<li>PCR of RBS-<i>abrB</i> and P<sub>xyl</sub> and purification of these PCR products.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Purification of the <i>xylR</i> gene.</li><br />
<li>Miniprep of RBS-xylR and electrophoresis test → the ligation failed again...</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Tuesday, August 7th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>There is a little plasmid pHT304 S and pHT315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without <i>Spe</i>I site.</li><br />
<li>Transformation in NM522 and spreading on LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li><br />
<li>Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).</li><br />
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li><br />
<li>New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours at 37°C (efficiency of the lysostaphin in stationary phase ?) → the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>3A ligation : [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + [psB1T3].</li><br />
<li>Transformation of the ligation [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + pSB1T3 in NM522 strain and spreading LB+Tet plates.</li><br />
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li><br />
<li>Purification of P<sub>xyl</sub> produced by PCR.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Wednesday, August 8th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 by pHT304 S and pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the <i>Spe</i>I site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...</li><br />
<li>Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.</li><br />
<li>New ligations : <br />
<ul><br />
<li>Promoter+Dispersin in pSB1C3;</li><br />
<li>Lysostaphin+Dispersin in pSB1C3.</li><br />
</ul><br />
Then transformation in NM522.</li><br />
<li>Plasmidic extraction of the BK12 strain : plasmid okay.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Results of the transformations :<br />
<ul><br />
<li>Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li><br />
<li>Transformation by <i>sfp</i>-<i>abrB</i>-pSB1T3 : the negative control isn’t good, the transformation is done again...</li><br />
</ul><br />
<li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Thursday, August 9th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Extraction of NM522/pHT315S. We expected a plasmid without a SpeI site. However, the selected clone contained a plasmid identical to pHT315 (the digestion by <i>Spe</i>I and <i>Eco</i>RI enzymes gives a fragment at 1000 bp). We decided to screen more clones (8 out of 17).</li><br />
<li>The transformation pSB1C3 + RFP and pSB1T3 + RFP is a success ! (red clones)</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
Transformation results : <br />
<ul><br />
<li>Positive control : okay;</li><br />
<li>Negative control : okay;</li><br />
<li>[Lysostaphin + Dispersin] : 8 clones;</li><br />
<li>[Promoter + Dispersin] : more than 30 clones.</li><br />
</ul><br />
Clones are screened on LB + Amp and LB + Cm plates.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The transformation of NM522 strain by <i>sfp</i>-<i>abrB</i> didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the <i>sfp</i>-<i>abrB</i> ligation again...</li><br />
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li><br />
<li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Friday, August 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Deletion of the <i>Spe</i>I site from pHT315 and pHT304 plasmids and filling-in using the <i>Pfu</i> polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li><br />
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.</li><br />
<li>None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.</li><br />
<li>Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.</li><br />
<li>Plasmidic extractions of these clones : clones not okay.</li><br />
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.</li><br />
<li>The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li><br />
<li>The NM522 strain with <i>abrB</i>-pSB1K3 was put in storage under the reference BK25.</li><br />
<li>The NM522 strain with <i>sfp</i>-pSB1K3 was put in storage under the reference BK26.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Saturday, August 11th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Gel electrophoresis showed that the elimination of the <i>Spe</i>I site was not successful.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Sunday, August 12th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Culture of 60 mL of Bs 168 pWG 100 at 28°C for the lysostaphin tests on plates.<br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Monday, August 13th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>We identified the reason why the deletion of the site <i>Spe</i>I from pHT315 and pHT304 plasmids and filling-in was not successful : the <i>Pfu</i> enzyme requires Mg<sup>2+</sup> and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li><br />
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth. These flasks are then frozen at -80°C and freeze-dried overnight.</li><br />
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligation [<i>sfp</i> + <i>abrB</i> + pSB1A3] in the NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Tuesday, August 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The gel electrophoresis of the digested plasmids pHT315 and pHT304 after the final purification shows promising results, so the two plasmids were transformed in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li><br />
<li>Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformation of the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Standard ligation between pBK7 (=RBS in pSB1C3) and xylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain. <br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Wednesday, August 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.</li><br />
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
None of the 4 clones tested had the expected fragments, so we screened 6 others.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by pBK7 and <i>xylR</i> : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + <i>xylR</i> contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and <i>xylR</i> (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Thursday, August 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones having integrated the modified shuttle vector (after filling-in). 4 clones seem to have the right plasmid.</li><br />
<li>The electrophoresis after digestion with <I>Eco</i>RI and <i>Spe</i>I showed that the <i>Spe</i>I site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive promoter (P<sub>veg</sub>) produced by PCR in an iGEM plasmid.</li><br />
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).</li><br />
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.</li><br />
<li>Ligation of P<sub>xyl</sub> produced by PCR in an iGEM plasmid.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Ligation of <i>xylR</i> produced by PCR in an iGEM plasmid (pSB1K3).</li><br />
<li>Results of the transformation of NM522 by [pBK7 + <i>xylR</i>] (for the second ligation) : the plate with bacteria transformed by [pBK7 + <i>xylR</i>] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + <i>xylR</i>]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without <i>xylR</i>.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Friday, August 17th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time, the digestion was successful. The tests showed that there was no <i>SpeI</i> site. However, the pHT315 plasmid also had no XbaI site. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a <i>XbaI</i> site. The digestion of the control plasmid was partial.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Plasmid extractions from the 14 clones transformed by [pBK7 + <i>xylR</i>] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without <i>xylR</i>.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Monday, August 20th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>pBK19 with no SpeI site was put in storage under the reference pBK25.</li><br />
<li>pBK20 with no SpeI site was put in storage under the reference pBK26.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Launch of 8 [Promoter+Dispersin] cultures.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + P<sub>xyl</sub>] : the plate with bacteria transformed by [pSB1T3 + P<sub>xyl</sub>] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + RBS-<i>abrB</i>] : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by [pSB1K3 + <i>xylR</i>] : the plate with bacteria transformed by [pSB1K3 + <i>xylR</i>] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without <i>xylR</i>.</li.<br />
<li>A new PCR of <i>xylR</i> is made, in order to increase the stock.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="21"><br />
<date>Tuesday, August 21st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector <i>E. coli</i> - <i>B. subtilis</i>). Different ligations are made with different proportions of insert and vector. Transformation in NM522 strain.</li><br />
<li>Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.</li><br />
<li>Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li><br />
<li>Results of the second transformation of NM522 by pSB1T3 and RBS-<i>abrB</i> : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Two standard ligations are done :</li><br />
<ul> <br />
<li>pBK7-<i>xylR</i> (<i>xylR</i> cut in X and P sites);</li><br />
<li>pBK24-<i>xylR</i> (<i>xylR</i> cut in E and S sites).</li><br />
</ul><br />
<li>Transformation of pBK7-<i>xylR</i> into NM522 strain.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Wednesday, August 22nd 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 with [Constitutive Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.</li.<br />
<li>New trial of cloning : Digestion, Ligation, transformation to construct :</li><br />
<ul><br />
<li>[Lysostaphin + Dispersin] in pSB1C3;</li><br />
<li>[Lysostaphin + Dispersin] in the shuttle vector (vector for <i>E. coli</i> and <i>B. subtilis,</i>).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 P<sub>xyl</sub> clones were tested, but none had integrated the plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Verification of 6 [<i>xylR</i>-pSB1K3] clones : they only have the vector.</li><br />
<li>Transformation of [pBK24-<i>xylR</i>] into NM522 strain.</li><br />
<li>Results of [pBK7-<i>xylR</i>] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li><br />
<li>Liquid culture of <i>B. subtilis</i> <i>abrB</i> in 5mL of LB broth.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Thursday, August 23rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformations results : All controls are okay.</li><br />
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li><br />
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li><br />
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)</li><br />
<ul><br />
<li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li><br />
<li>The second clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2);</li><br />
<li>The third clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of [P<sub>xyl</sub>+pSB1T3] in NM522.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of [pBK24-<i>xylR</i>] transformation : most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.</li><br />
<li>Extraction of pBK7-<i>xylR</i> from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> <i>abrB</i> in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate').<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Friday, August 24th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A ligation of pBK4 (pUC57 containing the <i>lacI</i> gene) and pBK29 (pSB1A3 containing the <i>sfp</i> and <i>abrB</i> genes) in pBK24 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>After the bad results with <i>xylR</i>, we decided to cut pBK10 plasmid in <i>Sma</i>I site, and ligate with <i>xylR</i>, doing a blunt ligation. We also decided to ligate <i>xylR</i> with <i>xylR</i>, creating a big polymer, which will be like a “pre-ligation” molecule.</li><br />
<li>Extraction of pBK24-<i>xylR</i> from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! Meaning that it’s the first time we manage to ligate <i>xylR</i> successfully.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> <i>abrB</i> strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms.<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date>Saturday, August 25th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of <i>Spe</i>I enzyme, so we could not digest the extracted plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
6 white clones from the ligation pBK24-<i>xylR</i> are purified and put in liquid cultures.<br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date>Sunday, August 26th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Extraction from the 6 white clones (containing <i>xylR</i>-pBK26). Gel electrophoresis is run, but it didn’t show any good clone. <br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date>Monday, August 27th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li><br />
<li>Midiprep : Extraction of P<sub>veg</sub>-dispersin-pSB1C3 of clone 22.</li><br />
<li>BK33 strain was put in storage.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li><br />
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P<sub>xyl</sub>, RBS and <i>sfp</i> gene), <i>abrB</i> gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
No transformation was done, because we ran out of LB broth. <br />
</description><br />
</jour><br />
<br />
<jour nb="28"><br />
<date>Tuesday, August 28th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Meeting at 1:30 pm.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li><br />
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria → Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br /><br />
We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid cultures and then streaked on LB+Cm plates and LB+Amp plates.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Transformation of <i>xylR</i>-pBK10 (blunt end ligation) in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="29"><br />
<date>Wednesday, August 29th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li><br />
<li>Results of the second transformation of Bs 168 strain with pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).</li><br />
<li>We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.</li><br />
<li>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> genes. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.</li><br />
<li>Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using two ligated genes (<i>sfp</i> and <i>abrB</i>) coming from clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classic protocol because we were running out of time and we were behind the schedule.</li><br />
<li>In parallel, we decided to do a standard ligation in order to transfer the <i>lacI</i> gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Transformation of <i>xylR</i>-pBK24 in NM522. The transformation of <i>xylR</i>-pBK24 is done in order to see if <i>xylR</i> was successfully ligated or not. If the bacteria are red, then <i>xylR</i> was not ligated.</li><br />
<li>24 clones containing pBK10-<i>xylR</i> are patched in LB+Amp growth medium, and then incubated.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date>Thursday, August 30th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li><br />
<li>12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.</li><br />
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li><br />
<li>Standard ligation between the shuttle vector pBK26 (pHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.</li><br />
<li>The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.</li><br />
<li>The miniprep confirmed that the ligation containing <i>lacI</i> was successful.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing <i>xylR</i>-pBK10 ligation. The clone is put in liquid culture overnight.</li><br />
<li>Another test is run to confirm that pBK34 plasmid (<i>xylR</i>-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since <i>xylR</i> contains a <i>Nde</i>I restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains <i>xylR</i>. Result : pBK34 doesn’t contain xylem.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date>Friday, August 31st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by <i>Spe</i>I, so the ligation was successful. The four plasmids are further tested for the other 3 iGEM sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digestion was done (<i>Eco</i>RI and <i>Spe</i>I). There was no fragment at 1,000 bp, so the ligation was successful.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li><br />
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li><br />
<li>Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :</li><br />
<ul> <br />
<li>The negative control is ok;</li><br />
<li>The positive control is full of colonies;</li><br />
<li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA. </li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the saturated cultures with transformed bacteria containing the ligation of the <i>sfp</i> and <i>abrB</i> genes. The electrophoresis confirmed that this time the bacteria contained the right plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.</li><br />
<li>The plasmid is digested by <i>Eco</i>RI, <i>Xba</i>I, <i>Spe</i>I and <i>Pst</i>I to find out if the problem comes from a bad site sequence. Result : the <i>Eco</i>RI restriction site is not good, therefore is not recognized by <i>Eco</i>RI enzyme.</li><br />
</ul><br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="September" size="30"><br />
<jour nb="1"><br />
<date>Saturday, September 1st 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the manufacturer protocol and another trial is made with higher concentration.</li><br />
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li><br />
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li><br />
<li>Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into <i>E. coli</i> NM522 strain.</li><br />
<li>6 other clones with the transformed 3A ligation (done on August 29th) are screened.</li><br />
<li>Standard ligation of the <i>abrB</i> gene and the plasmid containing <i>lacI</i> in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="2"><br />
<date>Sunday, September 2nd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated overnight.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized, followed by transformation in <i>E. coli</i> NM522 strain.</li><br />
<li>Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.</li><br />
<li>12 clones for each ligation (done the day before) are screened.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date>Monday, September 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Midiprep of the cloned shuttle vectors (containing the iGEM linker).</li><br />
<li>Miniprep of pBK26 shuttle vector is done in order to increase the stock.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>No clones are obtained on the plate lysostaphin+dispersin when we followed the manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.</li><br />
<li>Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.</li><br />
<li>A transformation is made with our cells of their positive control.</li><br />
<li>Transformations of Bs 168 :<br />
<ul><br />
<li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li><br />
<li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li><br />
</ul><br />
The bacteria are spread on LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).<br /><br />
The transformation of NM522 with ligation Dispersin in pBK26 was successful.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li><br />
<li>6 clones transformed with the mixture of two plasmids are screened.</li><br />
<li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the <i>lacI</i> and <i>abrB</i> genes). The clone is put in storage under the reference pBK39.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date>Tuesday, September 4th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 1.3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.</li><br />
<li>Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.</li><br />
<li>Results of the transformations of Bs 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li><br />
<li>Standard ligations between :<br />
<ul><br />
<li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Pst</i>I;</li><br />
<li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Eco</i>RI.</li><br />
</ul><br />
These two ligation products are transformed in NM522 strain.</li><br />
<li>Acrylamide gels and samples are prepared for SDS-PAGE.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis showed that 5 out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with <i>Eco</i>RV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A PCR is run in order to get RBS-<i>xylR</i>. The PCR failed (not enough DNA-polymerase).<br />
</description><br />
<titre>Biofilm</titre><br />
<description><br />
<ul><br />
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li><br />
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li><br />
<li>Same plate as the one above + crystal violet.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date>Wednesday, September 5th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK22 (<i>sfp</i> gene) and pBK39 (<i>abrB</i>-<i>lacI</i>) and transformation into the <i>E. coli</i> NM522 strain.</li><br />
<li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date>Thursday, September 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD<sub>600</sub> of the liquid cultures is. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Clones obtained on lysostaphin-dispersin plates are put in liquid culture.</li><br />
<li>Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD<sub>600</sub>600 measurements in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The Bs 168 strain is transformed by :<br />
<ul><br />
<li>pBK 25 (= modified shuttle vector pHT304);</li><br />
<li>pBK 26 (= modified shuttle vector pHT315);</li><br />
<li>pBK 28 (= Lysostaphin in the modified shuttle vector pHT304);</li><br />
<li>pBK 38 (= Lysostaphin in the modified shuttle vector pHT315);</li><br />
<li>Dispersin in pBK26.</li><br />
</ul><br />
We spread 200 µL of each transformed cells on LB + Erythromycin ([Ery] = 16 µg/mL) plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones transformed with the ligation pBK22 and pBK39 are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
2 new PCR are done to have <i>xylR</i>, but they failed. <br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Friday, September 7th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.</li><br />
<li>Result of the transformation of Bs 168 : </li><br />
<ul><br />
<li>All the negative control plates are clear : the erythromycin concentration is high enough to do a selection;</li><br />
<li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to extract and analyze their DNA;</li><br />
</ul><br />
<li>Transformation of Bs 168 : new trial by electroporation. As the last transformation, the Bs 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li><br />
<li>A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (<i>sfp</i>, <i>abrB</i> and <i>lacI</i>) would be higher.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A new RBS-<i>xylR</i> PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Saturday, September 8th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Miniprep of the clones Bs 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for <i>E.coli</i> with addition of Lysozyme to the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel → Maybe the DNA isn’t concentrated enough ?<br /><br />
New clones are put in liquid cultures in order to make other minipreps. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of P<sub>lac</sub>-RBS-<i>xylR</i> and then transformation in NM522.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Each of the 24 wells of two microtiter plates are inoculated with 2mL of a suspension obtained from diluting the liquid culture 100 times with TSB enriched with 1% glucose The plate is incubated during 24 hours at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Sunday, September 9th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones Bs 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li><br />
<ul><br />
<li>Bs 168 clone transformed by pBK25 has the right plasmid !! =)</li><br />
<li>Bs 168 clone transformed by pBK28 has the right plasmid !! <strong>BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</strong></li><br />
<li>Bs 168 clones transformed by pBK26 and dispersin in pBK26 : nothing on the gel... </li><br />
</ul><br />
<li>Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + Ery plates, with two different concentrations : [Ery]=10 µg/mL and [Ery]=15 µg/mL.</li><br />
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li><br />
<li>Thanks to the good results, we put in storage the transformed <i>Bacillus</i> under the reference :</li><br />
<ul><br />
<li>BK41 : Bs 168 + pBK25</li><br />
<li>BK42 : Bs 168 + pBK28</li><br />
<li>BK43 : Bs 168 + pBK26 (to confirm)</li><br />
<li>BK44 : Bs 168 + Dispersin in pBK26 (to confirm)</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The supernatant from the two microtiter plates are decanted. The wells of a third microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the dispersin. The supernatants are diluted in order to test different concentrations of the supernatants. The wells of a fourth microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the lysostaphin.<br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Monday, September 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.</li><br />
<li>Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the <i>E. coli</i> NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li><i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.</li><br />
<li>According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD<sub>600</sub> of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD<sub>600</sub>.</li><br />
<li>New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.</li><br />
</ul><br />
</description><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
<ul><br />
<li>Measurement of the NM522 + P<sub>lac</sub> kinetics (with Cm) during 12h in a 96 well plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)</li><br />
<li>In the same 96 well plate NM522 + P<sub>xyl</sub> kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)</li><br />
<li>Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plates prepared the day before are analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can see that the biofilm is not adherent enough and it is destroyed no matter what kind of supernatant is tested. Unfortunately, we cannot say what the effect of the dispersin or lysostaphin is using this kind of test.<br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Tuesday, September 11th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm (plate 2).</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).</li><br />
<li>Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones per transformation are screened.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. <i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.</li><br />
<li>SDS-PAGE is done using pellets, to find a good concentration.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Wednesday, September 12th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation. Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.</li><br />
<li>Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately, the screened clones did not integrate the expected plasmid.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture with the new protocol. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. There is a problem with the results : the control is not good.</li><br />
<li>Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>5 test tubes containing a lamella are inoculated with the culture prepared the day (<i>S. epidermidis</i>) before diluted 1:100 with TSB supplemented with 1% glucose.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Thursday, September 13th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding of 2 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>OD<sub>600</sub> measurements with filtered supernatants.</li><br />
<li>Mobility test of Bs168 on agar 0.25% plate.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.</li><br />
<li>Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Another 12 clones per transformation were screened (from the transformation made on 10th September).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in tanks to test dispersin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce dispersin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. Dispersin has no effect on <i>S. epidermidis</i> culture according to the results.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>One of the tubes and lamella prepared the day before containing <i>S. epidermidis</i> cultures is quantified using crystal violet staining and OD<sub>600</sub> is measured. <i>S. epidermidis</i> cells are quite adherent on glass surfaces.</li><br />
<li>The supernatant of the four other tubes are decanted and tubes are filled with the supernatants produced by the four liquid cultures of <i>B. subtilis</i> prepared the day before in order to test the effect of lysostaphin and dispersin.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Friday, September 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).<br /><br />
Moreover, OD<sub>600</sub> of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.</li><br />
<li>TG1 (<i>E. coli</i>) strain containing pJIM2241 shuttle vector is put in storage (BK47).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600s</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. The strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li>SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The four tubes and lamella prepared the day before are quantified using crystal violet staining and OD<sub>600</sub>is measured. The same result is obtained : <i>S. epidermidis</i> biofilms are not adherent enough and biofilms are destroyed when the growth medium is changed.<br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Saturday, September 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the 2 shuttle vectors pBK35 and pBK36.<br />
</description><br />
<br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in 96 well plate to test impact of lysostaphin and dispersin on a culture of <i>S. epidermidis</i> (test during 12 hours).<br />
</description><br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
LB plates containing a 0.3% agarose gel are inoculated with 5 μL of a 5 hour liquid culture of <i>B. subtilis</i> containing the plasmids used in transformations.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligations between the plasmid pBK42 containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] and the shuttle vectors pBK35 and pBK36 into the NM522 strain .<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Sunday, September 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Screening of 12 clones per <i>Bacillus</i> transformation.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 15 clones transformed with the ligation pBK42+pBK35 and pBK42+pBK36<br />
</description><br />
<br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
The diameter of the disks formed by <i>B. subtilis</i> is measured. The fact of introducing the plasmids did not impact the swarming mobility of our bacteria.<br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Monday, September 17th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Erythromycin at different concentrations was spread on LB plates with in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="18"><br />
<date>Tuesday, September 18th 2012</date><br />
titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened.<br />
</description><br />
<br />
<br />
</jour><br />
<br />
<jour nb="19"><br />
<date>Wednesday, September 19th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.<br />
</description><br />
<br />
<br />
<br />
<br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Thursday, September 20th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector. <br />
</description><br />
<br />
<br />
</jour><br />
<br />
<br />
<br />
<jour nb="21"><br />
<date>Friday, September 21st 2012</date><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
A 7-hour-long kinetics is made with P<sub>lac</sub> and P<sub>xyl</sub> to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used.<br />
P<sub>lac</sub> = IPTG 0.5% and P<sub>xyl</sub> = xylose 0.5%.<br /><br />
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
SDS-PAGE is done in order to detect Dispersin in <i>B. subtilis</i> and <i>E. coli</i>'s supernatants. The gels were not conclusive, and were not good due to an ammonium persulfate problem.<br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Saturday, September 22th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
12 clones transformed with the shuttle vectors are screened.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A new SDS-PAGE is done for dispersin. A migration problem persists, due to ammonium persulfate. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
24 clones transformed with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened.<br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Sunday, September 23th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing 2 mL liquid LB medium are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A last SDS-PAGE is run. This time the problems were successfully solved. But the gels were not conclusive due to a low dispersin concentration.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Monday, September 24th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Both strains grew in all tubes. However, there is a difference concerning OD<sub>600</sub> between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1.5 mg/mL) in order to make sure there is a notable difference between the two strains. </li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<li> In order to characterize the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] two tests are made. To confirm the presence of <i>sfp</i> gene, we made emulsions with the filtered supernatant of the transformed strains and sunflower oil. Concerning <i>abrB</i> gene, a biofilm formation test was made in a 24-well microplate in order to compare the transformed strain to the wild-type strain. <br />
</li><br />
</description><br />
</jour><br />
<jour nb="25"><br />
<date>Tuesday, September 25th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes; <br />
</description><br />
</jour><br />
<br />
</month><br />
<br />
<month name="October" size="31"><br />
<br />
</month><br />
<br />
</project><br />
</xml><br />
</div><br />
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<h1>Our collections</h1><br />
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<div class="unProto" onclick="window.open('https://static.igem.org/mediawiki/2012/8/89/Tableau_souches.pdf', 'Strains collection'); return false;">Strains collection</div><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/sponsorsTeam:Lyon-INSA/sponsors2012-10-16T18:24:34Z<p>Suxiaohui: </p>
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/File:Logo_Ambassade_Etats-Unis.jpgFile:Logo Ambassade Etats-Unis.jpg2012-10-16T18:24:07Z<p>Suxiaohui: </p>
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<div></div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/videosTeam:Lyon-INSA/videos2012-09-27T03:44:56Z<p>Suxiaohui: </p>
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<br><center>We are glad to present you the Lyon-INSA 2012 iGEM team.</center> <br><br><br />
<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtwcrk?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtwcrk_lyon-insa-2012-igem-team_tech" target="_blank">Lyon INSA 2012 iGEM team</a> </center><br><br><br />
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<center>This 2012 iGEM competition was a very amazing experience, and we would like to share our journey with you.</center><br><br><br />
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<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtwcp8?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtwcp8_mon-film-part2-fin_tech" target="_blank">Journey of Lyon INSA 2012</a></center><br><br><br />
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<center>If you haven't already seen the project description, this is how Bacillix is working !</center><br><br><br />
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<center><center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtsdia?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtsdia_our-project-presentation-biofilm-killer-lyon-insa-igem_tech" target="_blank"><font color ="purple">Our project presentation Biofilm Killer Lyon...</font></a></center></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/collaborationTeam:Lyon-INSA/collaboration2012-09-27T03:43:55Z<p>Suxiaohui: </p>
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<h1>How did we collaborate?</h1><br />
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Collaboration is a very important issue. We think that sharing our ideas could be very beneficial for both teams engaged in the collaboration. There are more than 240 teams involved in the iGEM 2012 so it was impossible not to find another team sharing some aspects of our project.<br><br />
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<h3>iGEM FRANCE</h3><br />
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<a href="http://sites.google.com/site/igemfrance/"><font>iGEM FRANCE</font></a> is a collaborative website that gathers the french iGEM community together. It has been created in the beginning of 2012 by Paris-Bettencourt team as a help for students and professors to create and run their iGEM team. The website is above all a way to share knowledge, either through its forum, or by arranging meetings. <br><br />
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<h3>Collaboration with the University of British Columbia (UBC)</h3><br />
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We have established a collaboration with <a href="https://2012.igem.org/Team:British_Columbia">UBC</a> for our human practices. Indeed UBC is working on Intellectual property (IP) this year, an aspect we also chose to explore this year.<br><br />
One of our advisors is a Professor in Economics. Thus, we have suggested to UBC when they sent their survey on IP, that we could share with them our knowledge and thoughts to improve their survey or to interpret the results of their survey. In exchange they shared the results of their survey with us.<br><br />
We have used their results to show what kind of IP issues iGEM teams could meet.<br />
To see the results of our collaboration you might want to read our <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><font> human practice </font></a>.<br><br />
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<h3>An interesting way to undertake a collaboration with Göttingen team</h3><br />
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Furthermore, we have tried to begin a collaboration with the <a href="https://2012.igem.org/Team:Goettingen">Göttingen team</a>. They are working on a super swimmer <i>E. coli</i> strain. We have proposed them different ways for collaboration. For example, we were interested in comparing the impact of swimming on biofilm destabilization between their <i>E. coli</i> strain and our <i>Bacillus subtilis</i> natural swimmer strain. We offered to send them our <i>Bacillus subtilis</i> strain to also make them able to perform comparative swimming behavior studies between both strains. Several different reasons made this collaboration unsuccessful.<br><br />
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<h3>Collaboration with TU Munich</h3><br />
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We took part in <a href="https://2012.igem.org/Team:TU_Munich">TU Munich</a>'s survey about their proposition of standardization of BioBricks part descriptions.<br><br />
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<center><img id="TUM12_badge" width="150px" src="https://static.igem.org/mediawiki/2012/c/c6/TUM12_Collaboration_medal1.png" alt="We completed TU Munich's survey on Standardization of BioBrick part descriptions"/></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/townTeam:Lyon-INSA/town2012-09-27T03:41:18Z<p>Suxiaohui: </p>
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<h1>Our town : Villeurbanne</h1><br />
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<div class="introduction contenuTexte" style="margin:20px;display:inline-block;width:60%;"><br />
<p> The Lyon-INSA iGEM team is not actually in Lyon but in Villeurbanne, which is a city on the outskirts of Lyon. In fact, the school is located on the Doua campus of Villeurbanne.<br />
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The city counts about 144 000 inhabitants distributed on an area of approximately 15 km&sup2;. Historically a workers' suburb of Lyon, Villeurbanne is nowadays an academic and working class city.<br />
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But let us introduce our wonderful city :<br />
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<img src="https://static.igem.org/mediawiki/2012/c/c8/7746272115_villeurbanne-dans-la-banlieue-de-lyon.jpg" width="30%" style="border:5px solid white;<br />
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<h2>The history of Villeurbanne</h2><br />
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<a href="https://static.igem.org/mediawiki/2012/c/c7/Satellite.jpg" class="fancyable"><br />
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Villeurbanne joined the France Kingdom in 1355. The expansion of the city was limited by the frequent floods due to the Rhone river until 1837, when dykes started being used as water defences. Starting with the 1789 Revolution, the city was integrated to the department of Isere and joined the Rhone department only in 1852. At that time, the city refused its unification with the city of Lyon but was forced to give away a part of the famous "Parc de la Tete d'Or".<br />
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During the Industrial European Revolution of the XIXth century, Villeurbanne knew a quick industrial development. However, when World War I broke out, the working force went missing, so women and immigrants were called in to fill in the positions not only in the city, but in the whole country.<br />
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During World War II, Villeurbanne and Lyon were a strategic area. Lyon was situated on the borderline between Nazi-occupied area and unoccupied area by Germany. Lyon and Villeurbanne provided a home for great figures of the French resistance and, above all, provided the headquarters for the printing of resistance newspapers. A lot of the resistance fighters were caught in Lyon due to their activity in the printing of newspapers and were killed in Villeurbanne, on the current campus, which was at that time a military area.<br />
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In August 1944 the city was freed by an insurrection. Nonetheless, 3 days later, the German took the city back but it was eventually liberated by the troops of the Provence landings a couple of days later. <br />
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Since 1960, the factories and small properties began being replaced by buildings hosting the middle class.<br />
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<h2>Monuments and culture</h2><br />
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<h3>Festivals and events</h3><br />
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<div class="contenuTexte" style="width:60%;display:inline-block;"><br />
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Throughout the year lots of events are organised in Villeurbanne. For example, in Mars there is an Iberian and Latino-american film festival called "Refets du Cin&eacute;ma ib&eacute;rique et latino-am&eacute;ricain" and for the sportive ones a foot race.<br />
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In may, the "24 heures de l'INSA de Lyon" is an event not to be missed under any circumstances! For a all night and day week end a lot of free different activities are proposed : concerts, funny games, 24 hour long races, dances, massage, board games and even bungee jumping. Moreover, anyone between 7 and 77 can participate!<br />
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In November, when the cold comes, you can go to the Short Film Festival and enjoy the movies !<br />
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<a href="https://static.igem.org/mediawiki/2012/a/a9/0._les_24h%2Bserpe.jpg" class="fancyable"><br />
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<h3>Monuments</h3><br />
<div class="petitSsTitre">TNP</div><br />
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A little story..<br />
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The Th&eacute;&acirc;tre National Populaire (TNP), National People's Theatre, was founded in 1920 by Firmin G&eacute;mier in Paris. It has been located in Lazard-Goujour square in the Villeurbanne's Skyscrapers district since 1972, instead of the city's Theatre.<br />
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This dramatic national center managed to implant in the region of Lyon a standing creation theatre. We remember the national and international theatre tours of the Three Musketeers, Tartuffe, Henry IV...<br />
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It has certainly contributed to the creation, but also to the production and the hosting of other plays that are performed all around France. Besides, it has its own set construction workshop. It is one of the first theatres with its own development strategy, making it a real "theater company". Even though with tickets at affordable prices, attracting at least 2500 people every night is a real challenge.<br />
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Indeed, the primary goal of the TNP is to offer quality plays, accessible to as many people as possible. This means meeting directly public's expectations by developing a special communication policy.<br />
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The TNP is a symbolic theater, symbolic by its acronym, by its act, by its history and by the men who have imposed it.<br />
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<div class="petitSsTitre">Media library</div><br />
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<div class="contenuTexte" style="width:60%;display:inline-block;"><br />
In the late '80s, a media library, called "Maison du livre, de l'image et du son", was built according to Mario Botta's plans.<br />
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<div class="petitSsTitre">The Rize</div><br />
<a href="https://static.igem.org/mediawiki/2012/7/7b/3.Le_rize%2Basterix.png" class="fancyable"><br />
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The Rize, a research and cultural museum, opened its doors in 2008. It contains a representative collection of municipal files, a media library and provides spaces for cultural and educational activities.<br />
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<div class="petitSsTitre">The skycrapers ("les gratte-ciel")</div><br />
<div class="contenuTexte" style="width:50%;display:inline-block;margin-top:10px;"><br />
Built during the early 30s, "The Skycrapers" stand up amongst fields strewed with houses and factories. It is a neighborhood with giant vertical buildings, a very peculiar architecture for a population used to buildings with less than five floors.<br />
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Meant to shelter social and workers' housing, these skyscrapers were inspired by several European and North American influences. In fact, the agglomeration of Lyon had successful economic relationships with the North-Eastern industrial regions of the USA at the end of the XIXth century. <br />
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Being the first European construction of residential buildings of this height, it captivated the specialists at that time. Moreover, its construction during the Great Depression makes it even more unique.<br />
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However, this new modern district (and even futurist at that time) is not only a architectural curiosity, but also an assertion of the identity of Villeurbanne. Synonymous of social breakthrough and political affirmation, the Skycrapers district represents for the population the true symbolic center of the city. It shows that Villeurbanne is a separate town from Lyon and finally ends the intents of geopolitical attachment between the two cities. <br />
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<div class="petitSsTitre">Modern Art</div><br />
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If you have an artistic spirit or if you just want to see how modern art looks like, Villeurbanne and its Institute and Museum of Contemporary Art is made for you. Villeurbanne has also a Metropolitain Center of Urban Art since 2002 : "Les Ateliers Frappaz". In collaboration with the city's cultural departement, the Museum organises in mid-June a free music and street art festival called "Les Invites de Villeurbanne".<br />
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<div class="petitSsTitre">Sport</div><br />
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As you can imagine, Villeurbanne is not only a cultural city but also a sportive one. As the latin saying goes, "Mens sana in corpore sano" ("a sound mind in a healthy body"),Villeurbanne inhabitants can use both brains and body.<br />
<br/><br />
In order to illustrate Villeurbanne has nighty-nine stadiums, thirteen sport rooms, four areas for playing boules, three swimming pools, five climbing walls ande more than two hundred sports clubs. One of the most important and perhaps the best known is the ASVEL club (l'Association sportive de Villeurbanne &Eacute;veil lyonnais), especially its basket section.<br />
<br/><br />
Founded in 1948, the Basketball club of ASVEL competes in Pro A, the elite French championships. It is the French club with the most enviable prize list : it has been champion of France 17 times. <br />
<br/><br />
The ASVEL has several great players. The best known are Alain Gilles (from 1965 to 1992) and Delaney Rudd (from 1995 to 2001). In 2009, the famous NBA player in the France team Tony Parker was signed to the club of Villeurbanne. Moreover, he is the newly appointed director in charge of basketball operations.<br />
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<h2>Campus de la Doua</h2><br />
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<div class="introduction"><br />
La Doua is an ancient word used to designate a basin, a fountain, a wash house or a small watercourse. <br />
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<h3>History</h3><br />
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<a href="https://static.igem.org/mediawiki/2012/5/50/Cimetiere_militaire%2Bidefix.jpg" class="fancyable"><br />
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The campus of La Doua was created in 1957 and it covers 100 acres.<br />
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The place used to house a military training camp named Grand Camp which took the place's name and became the Doua barrack. For many years during the XIXth century it hosted the cavalry before the transition to the armored army.<br />
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During the German occupation of the Second World War, it was used as an execution camp. In September 1945, 77 resistant fighters' corpses were founded near a knoll known today as "the shot knoll". In 1957 the national necropolis of La Doua was built around the campus. It is a military cemetery, which is still standing nowadays in front of the INSA's buildings.<br />
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<h3>INSA</h3><br />
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Founded in 1957 by Gaston Berger and the rector Capelle, INSA was at first a school meant for senior engineers and technicians, but it quickly became an engineering school only.<br />
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Its uniqueness was to enroll student straight after their Baccalaur&eacute;at (equivalent of A level) and not after a two-year-preparatory class like the other French engineering schools.<br />
The purpose was to allow students from working and middle classes to do great studies at low cost.<br />
<br/><br />
Since the beginning, the school has been developed on a sharing and exchange policy. That's why it invests a lot in the boarding school and in the support of associations. Nowadays INSA counts more than 100 associations (sport, music, charity, culture,...).<br />
<br/><br />
The first promotion counted 300 students. Nowadays, it has more than 4500 engineering students of each 1400 graduate each year. INSA is no more a single school but a group of five schools located in Lyon, Strasbourg, Rennes and Rouen. However, Lyon is not only the oldest, but also the largest.<br />
<br/><br/><br />
The Lyon university is organised around 9 departements :<br/><br />
- Civil engineering and urbanism<br/><br />
- Biological engineering (subdivided in modelisation/ bioinformatics and biochemistry/ biotechnologies)<br/><br />
- Electrical engineering<br/><br />
- Computer sciences<br/><br />
- Material sciences and engineering<br/><br />
- Mechanical engineering<br/><br />
- Engineering systems and managements<br/><br />
- Telecommunications and network engineering<br/><br />
- Environmental and energy engineering<br/><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/collaborationTeam:Lyon-INSA/collaboration2012-09-27T03:40:24Z<p>Suxiaohui: </p>
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<h1>How did we collaborate?</h1><br />
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Collaboration is a very important issue. We think that sharing our ideas could be very beneficial for both teams engaged in the collaboration. There are more than 240 teams involved in the iGEM 2012 so it was impossible not to find another team sharing some aspects of our project.<br><br />
<br><br />
<br />
<h3>iGEM FRANCE</h3><br />
<br />
<a href="http://sites.google.com/site/igemfrance/"><font>iGEM FRANCE</font></a> is a collaborative website that gathers the french iGEM community together. It has been created in the beginning of 2012 by Paris-Bettencourt team as a help for students and professors to create and run their iGEM team. The website is above all a way to share knowledge, either through its forum, or by arranging meetings. <br><br />
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<h3>Collaboration with the University of British Columbia (UBC)</h3><br />
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We have established a collaboration with <a href="https://2012.igem.org/Team:British_Columbia">UBC</a> for our human practices. Indeed UBC is working on Intellectual property (IP) this year, an aspect we also chose to explore this year.<br><br />
One of our advisors is a Professor in Economics. Thus, we have suggested to UBC when they sent their survey on IP, that we could share with them our knowledge and thoughts to improve their survey or to interpret the results of their survey. In exchange they shared the results of their survey with us.<br><br />
We have used their results to show what kind of IP issues iGEM teams could meet.<br />
To see the results of our collaboration you might want to read our <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><font> human practice </font></a>.<br><br />
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<h3>An interesting way to undertake a collaboration with Göttingen team</h3><br />
<br />
Furthermore, we have tried to begin a collaboration with the <a href="https://2012.igem.org/Team:Goettingen">Göttingen team</a>. They are working on a super swimmer <i>E. coli</i> strain. We have proposed them different ways for collaboration. For example, we were interested in comparing the impact of swimming on biofilm destabilization between their <i>E. coli</i> strain and our <i>Bacillus subtilis</i> natural swimmer strain. We offered to send them our <i>Bacillus subtilis</i> strain to also make them able to perform comparative swimming behavior studies between both strains. Several different reasons made this collaboration unsuccessful.<br><br />
<br><br />
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<h3>Collaboration with Munich</h3><br />
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We took part in <a href="https://2012.igem.org/Team:TU_Munich">Munich</a>'s survey about their proposition of standardization of BioBricks part descriptions.<br><br />
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<center><img id="TUM12_badge" width="150px" src="https://static.igem.org/mediawiki/2012/c/c6/TUM12_Collaboration_medal1.png" alt="We completed TU Munich's survey on Standardization of BioBrick part descriptions"/></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/HumanPracticeTeam:Lyon-INSA/HumanPractice2012-09-27T03:35:25Z<p>Suxiaohui: </p>
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<div class="introduction contenuTexte" style="margin:20px;font-size:15px;"><br />
<p>Our human practice project includes two themes addressing many very different questions.<br />
The first ones are about various intellectual property issues. The other theme relates to the popularization of sciences and more specifically to our contribution to the researchers’ night.</p><br />
<p>We have chosen to divide the theme "intellectual property" into two parts because this theme is the main part of our reflexion and it required a longer development, but also because we collaborated with the <a href="https://2012.igem.org/Team:British_Columbia">University of British Columbia</a> on this theme and an entire part seemed necessary to explain our work.<br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and is made up by a reflexion on the economic challenges the synthetic biology industry faces. The second one sums up our collaborative work on the intellectual property with the UBC iGEM team. And the third one quickly presents our scientific popularization involvement. </p><br><br />
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<br />
<h2>Part I: Intellectual property issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
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<h3>Introduction</h3><br />
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Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements:iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
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<br><br />
Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
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The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Common's system", which will be discussed further on. We will assume the iGEM team members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
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<div class="introduction contenuTexte" style="display:inline-block;width:70%;"><br />
We will most notably refer to both Joseph Stiglitz’s (Nobel prize in economics in 2001) and Elinor Ostrom’s (Nobel prize in economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
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<h3>The patent system: an efficient economic model ?</h3><br />
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<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first one to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
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Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted invention have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
<br><br />
A patent is granted:<br><br />
<ul><br />
<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
</ul><br />
<br><br />
<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
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The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even the genetically modified bacterias may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 80s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy.).<br><br />
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The goal of this brief study though, is not to discuss this matter.<br><br />
<br><br />
<div class="petitSsTitre">Why patents have been created?</div><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new age for intellectual property began.<br><br />
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Instead, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
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Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patent’s creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
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As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
<br><br />
<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
<br><br />
<br><br />
For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components.When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
<br><br />
Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
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<br />
<div class="introduction contenuTexte" style="display:inline-block;width:65%;"><br />
As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licences of many expensive patents from the incumbent firms.<br><br />
<br><br />
<br><br />
Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief resume. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ?. Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such, e.g., as shown in inventions and discoveries of more and better ways and means, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
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This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
<br><br />
Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing.In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
<br><br />
<div style="margin:30px;"><br />
<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
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<br><br />
<br />
In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation.The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to embark on a collective reflexion on its contribution to the development of a "synthetic biology commonns”.<br />
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<h3>The Commons: an alternative model</h3><br />
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<br><br />
<br />
The open source software's users have been in ever greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies 92% used open source software during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
<br><br />
<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license.This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
<br />
<br><br />
<br><br />
The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
<br><br />
<br><br />
However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel prize in economics in 2009, broadened the economists' mind on this particular topic.<br />
<br><br />
<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th century.<br />
<br><br />
<br><br />
Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches: each community managing a commons fixes its own rules by defining a governance frameworkl and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
<br />
<br><br />
<br><br />
Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits.<br />
<br />
<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
<br />
<li>rules in use are well matched to local needs and conditions</li><br />
<br />
<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
<br />
<li>the right of community members to devise their own rules is respected by external authorities</li><br />
<br />
<li>a system for self-monitoring members’ behavior has been established</li> <br />
<br />
<li>a graduated system of sanctions is available</li> <br />
<br />
<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
<br><br />
<br><br />
<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
<br><br />
Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
<br><br />
The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic value of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
<br><br />
<br><br />
<br />
<h3>Bibliography</h3><br />
<br><br />
<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
<br />
<br />
</div><br />
</div><br />
<br />
<h2>Part II: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey.281 iGEMers had answered the survey when we treated it .<br />
<br><br />
This collaboration allowed us to broaden our reflexion to iGEM teams' issues.<br />
<br><br />
<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly we studied past iGEMers experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
<br><br />
Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
<br><br />
Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may results in a patent experience if the materials used are under a proprietary license. <br />
<br><br />
Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers IP experiences.<br />
<br/><br />
<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what was its nature. The results are that most of them (46 %) had already used patented materials before. However, 15% renounced to actually use them, which shows how patent can impede research and innovation: getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
<br><br />
The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10 % of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
<br><br />
Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
<br><br />
Our sole conclusion on this matter is that the IPR often does affect iGEM projects, even if in general the patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
<br />
<br><br />
It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
<br><br />
Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
<br><br />
To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
<br><br />
<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member or / and to a faculty advisor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
<br><br />
<br><br />
<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the intellectual property rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks .<br />
<br><br />
Besides, this survey deepened our main reflexion (q.v. part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the intellectual property rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
</div><br />
</div><br />
<h2>Part III: Scientific popularization</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br><br />
<br><br />
A lot of stereotypes and pieces of information circulate on sciences, so that we can sometimes find it hard to know where the truth is hidden. The public may sometimes look for a reliable source to make its own opinion. We are convinced that we have a role to play and have decided to involve ourselves in the researchers' night which is a european scientific popularization event.<br />
<br><br />
<br><br />
<h3>More about the researchers'night</h3><br />
<br><br />
<br><br />
Since 7 years, this event is taking place on a single September night in about 300 cities all over Europe. During the Researcher's night, everyone can come and explore science in engaging ways. The main goal of this night is to put researchers in touch with public concern, and explain what researchers are doing, how, and why it is important for everybody’s daily life.<br />
<br><br />
</ul><br />
<br><br />
This year, the theme is "Imagine the future": what future will emerge from research laboratories? How do the scientists imagine the future? Archeology, Physic, Phylososphy, Biosciences or Linguistic: researchers share their lab experiences and thoughts on the future.<br />
<br><br />
<br><br />
For the first time, the Lyon-INSA iGEM team is taking part to this event. We will discuss how synthetic biology can help in the future, but also what the potential negative impacts of this new discipline are. We would like people not to think to corn only when they hear the word "GMOs". To do so we decided to focus on medical application of GMO's as insulin, growth hormone, artemisine,...<br />
We have to be opened to all possible questions and be prepared to face a very heterogeneous public.<br />
<br><br />
<br><br />
<br />
<em>This event will take place on Friday night, 28 september 2012 at CCO Villeurbanne.<br />
That is why this part is so short. The wiki is freezing in a few hours from and we will describe our adventures later if we get to go to Boston. If you want to read it we will be there!</em><br />
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<div class="introduction contenuTexte" style="margin:20px;font-size:15px;"><br />
<p><br />
<em> Special thanks to our faculty advisor O. Brette for his advice and for his working late to help us and to the UBC team for sharing its poll results with ours. </em><br />
</p><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/HumanPracticeTeam:Lyon-INSA/HumanPractice2012-09-27T03:33:46Z<p>Suxiaohui: </p>
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<p>Our human practice project includes two themes addressing many very different questions.<br />
The first ones are about various intellectual property issues. The other theme relates to the popularization of sciences and more specifically to our contribution to the researchers’ night.</p><br />
<p>We have chosen to divide the theme "intellectual property" into two parts because this theme is the main part of our reflexion and it required a longer development, but also because we collaborated with the University of British Columbia on this theme and an entire part seemed necessary to explain our work.<br />
As a summary : the first part deals with the intellectual property rights inference towards the iGEM contest and is made up by a reflexion on the economic challenges the synthetic biology industry faces. The second one sums up our collaborative work on the intellectual property with the UBC iGEM team. And the third one quickly presents our scientific popularization involvement. </p><br><br />
<br><br />
<br />
<h2>Part I: Intellectual property issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
<br />
Free knowledge sharing is part of the foundational principles of the iGEM contest. Consequently, protecting the possible commercial uses of the iGEM team members’ projects seems difficultly feasible by common ways (especially patents and copyrights). However, the intellectual property rights in general and patents in particular are commonly known to be the only way to promote innovation in a profit-making logic: if companies cannot secure the economic outcome of their investments in R&D, they will not invest in it. A simple conclusion could then easily be drawn from these very statements:iGEM projects would be unlikely to be industrially and commercially developed, to the extent that it would be difficult for companies to capture the economic returns of their investments.<br />
<br><br />
<br><br />
Nonetheless, the decision of opening an iGEM entrepreneurship division, whose objectives are clearly to optimize the economic opportunities given by the performing iGEM innovation system, vigorously shook that very last conclusion.<br><br />
<br><br />
<br />
The aim of this human practice project is to show that patents are not the only way, not even always the best mean to promote innovation. Viable alternative models do exist, such as the "Common's system", which will be discussed further on. We will assume the iGEM team members form a commons similar to the Open Source community of the IT sector to strike up a reflexion on the development of alternate economic solutions to patents.<br><br />
<br><br />
<br />
<div class="introduction contenuTexte" style="display:inline-block;width:70%;"><br />
We will most notably refer to both Joseph Stiglitz’s (Nobel prize in economics in 2001) and Elinor Ostrom’s (Nobel prize in economics in 2009) theoretical work in the economics of innovation and intellectual property to back up our study.<br />
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<a href="https://static.igem.org/mediawiki/2012/4/4e/Patent.gif" class="fancyable"><br />
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<h3>The patent system: an efficient economic model ?</h3><br />
<br />
<div class="petitSsTitre">What is a patent, how is it awarded?</div><br />
A patent grants its owner a monopoly in a country on an invention he was the first one to describe and claim. At a firm's scale, this also means being able to shield the potential commercial exploitation of the R&D work which has led to the invention, against the company's competitors. It rewards this work by granting the patent’s owner an economic advantage that will probably make the financial cost of the R&D be worth it. The perspective of this heavy economic asset is the major reason why patents are said to be a driving force that encourage innovation.<br><br />
<br><br />
Nonetheless, such a profit-making privilege has a cost for both the claimer (patent offices fees, attorney fees, translation expenses if necessary, and so on) and the society, namely the "monopoly rent" it generates (see below). Indeed, targeted invention have to meet tough conditions for a patent to be granted. Consequently, knowing the granting conditions is very important to fully understand the patent system.<br><br />
<br><br />
A patent is granted:<br><br />
<ul><br />
<li>for a new invention never publicly revealed, which can lead to industrial applications ;</li><br />
<li>on a precise territory ;</li><br />
<li>for a maximum of twenty years ;</li><br />
<li>to the one who revealed the invention and described it with enough precision.</li><br />
</ul><br />
<br><br />
<i>NB : A patent has to be paid annually for its delivery and maintaining.</i><br><br />
<br><br />
The 1930 plant patent act marked the beginning of a new economic era for biology as emerged with it the patentability of the living world. Nowadays, even the genetically modified bacterias may be patentable (the latter have been patentable in the USA and then in Europe since the beginning of the 80s even if the precise conditions to be met to make "living things" patentable remain quite fuzzy.).<br><br />
<br> <br />
The goal of this brief study though, is not to discuss this matter.<br><br />
<br><br />
<div class="petitSsTitre">Why patents have been created?</div><br />
<a href="https://static.igem.org/mediawiki/2012/8/8f/Droits_homme.jpg" class="fancyable"><br />
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In France, patents have been created after the 1789 French Revolution. They were then considered as a human right : their aim was to recognize the inventors’ rights on their ingenuity. And so was born the first legal mean to claim authorship of an invention: a new age for intellectual property began.<br><br />
<br><br />
<br><br />
Instead, the justification for patents became quickly socio-economic. Patents are now conceived as an incentive for production and knowledge spreading. When a patent is awarded, the description of the invention goes public, in exchange for a maximum twenty-years commercial monopoly to the owner.<br />
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Patents are actually supposed to promote innovation and the disclosure of technological knowledge, which means allowing the latest inventions to be known by anyone, though their commercial use is forbidden without a license from the patent owner until the patent ends. On the one hand, the benefits that patents are expected to offer to society are very clear as their design is specifically supposed to improve the global technological level. Indeed, huge technological steps have been made since the patent’s creation (although it is not the only factor). On the other hand, stimulating the competition FOR the market (i.e for “new” markets) by granting some kind of monopoly rents generates economic costs for society, which derive from a weakening in the competition IN the market (i.e. in the “same” market).<br />
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As a result, the patent system rests on a socio-economic balance, in which society is supposed to be beneficiary. Nevertheless, as we will further see, this balance is fragile and depends on the answers to the following questions :<br><br />
<ul><br />
<li>What is the patentability scope ? What can be patented ? ;</li><br />
<li>How patents are granted ? (i.e. the toughness of the conditions required) ;</li><br />
<li>What are the rights of the patent owner ? ;</li><br />
<li>How long do these rights last ?</li> </ul><br />
<br><br />
<div class="petitSsTitre">Economic limits of the patent model</div><br />
Several factors (change in laws and in the practices of patent offices, technological and industrial evolution, etc.) may lead this balance between social costs and advantages to be broken. In some cases, the patent system may lead to hamper the innovation dynamics.<br />
<br><br />
<br><br />
For instance, narrow patents are sometimes granted to different companies on very specific elements, the gathering of which may be necessary to develop an innovation (a new product for example). Nowadays, major worldwide companies use patents as defensive or offensive tools to block competition by preventing (potential) rivals to use specific useful components.When powerful enough, these rivals strike back by doing the exact same thing on other components so that they are mutually blocked. This case is known as the "patent thicket" problem. As an example Google has recently bought Motorola Mobile and its 17000 patents for 12 billion dollars, which means they spent a lot of money to get these patents probably as offensive / defensive ends instead of spending it into pure R&D. Consequently patents sometimes paradoxically discourage innovation.<br><br />
<br><br />
Contrary to the previous case, some patents may protect wide discoveries (also known as foundational patents). For instance, the patent granted to Myriad genetics covered the whole gene functions of BRCA1 and BRCA2 and all applications that could follow on possible ways to diagnose and cure breast cancer. This kind of patent stood against innovation, preventing any creative use of those genes by any other actor than Myriad Genetics, or leading to any other use linked to this DNA sequence (possibly there would have been many, considering the complexity of biological regulations).<br><br />
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As a consequence, the patent system may allow the formation of major entry barriers on some industrial domains. It is notably the case in some markets of the pharmaceutical industry in which new companies cannot easily enter anymore because they would need to buy the licences of many expensive patents from the incumbent firms.<br><br />
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<br><br />
Last but not least, the philosophical questions raised in the last two centuries concerning patents are to be considered. As this is not a philosophical essay, we will content ourselves with a brief resume. According to the American economist Thorstein Veblen (1908), a patent is illegitimate as it privatizes a collective work any invention crucially depends on the previous ones : how would a trolley have been invented if the wheel had not been invented before ?. Veblen emphasizes this idea when he argues that:<br />
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<i>The initiative and technological enterprise of individuals, such, e.g., as shown in inventions and discoveries of more and better ways and means, proceeds on and enlarges the accumulated wisdom of the past. Individual initiative has no chance except on the ground afforded by the common stock, and the achievements of such initiative are of no effect except as accretions to the common stock. And the invention or discovery so achieved always embodies so much of what is already given that the creative contribution of the inventor or discoverer is trivial by comparison. (1908)</i><br><br />
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This thinking still applies nowadays on different themes. So the question remains : why should a sole actor gather all the economic benefits from this collective process?<br><br />
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Consequently, in some cases, having an economic alternative to the patent system may actually be a good thing.In this perspective the leading economists Claude Henry and Joseph Stiglitz assert that :<br><br />
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<i>The patent system is only one part of a society's innovation system, through which the production of knowledge is financed, incentivized and organized. Too much attention has been focused on IPR (intellectual property rights), and too little on alternatives, e.g. open source systems, publicly financed innovation and prizes. (2010)</i><br />
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In a recent paper, Claude Henry and Joseph Stiglitz (2010) argue that the open source system, which has been widely used in the IT sector, can be an alternate solution to the patent framework to promote innovation.The aim of our approach is not to precisely characterize the way the "open source approach" could be adapted to the synthetic biology. It is to urge the iGEM community to embark on a collective reflexion on its contribution to the development of a "synthetic biology commonns”.<br />
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<h3>The Commons: an alternative model</h3><br />
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The open source software's users have been in ever greater numbers for the last few years. According to a study made by Markess International in France in 2009, on 160 French companies 92% used open source software during that year. The viability of its mere founding principle explains its success : the involved software can be used freely with a license (“open source license”) authorizing it and even allowing its source code to be modified and enhanced by the licensed one. At first glance, such a model would seem to be inefficient as the software should not generate any profit : their license makes them indeed free to use. However, companies have opted for a business model involving services and proprietary software (“proprietary bricks”) that complement the open source product.<br />
<br>Besides, the mere nature of this emerging sector makes it very innovative : a whole community back it up by enhancing the software's source codes. This system also allows companies to make profit on a patent-free position. That is why such a successful example could serve as an inspiration for our reflexion.<br />
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<div class="petitSsTitre">From the open source software to the commons approach</div><br />
The open source system functions thanks to a wide community literally owning the diverse software under license.This community defines the rights and duties attached to the use and development of this software. As a consequence, the open source can be considered as a case of commons, i.e. a resource jointly owned by a group. This vague and ancient term has covered various kinds of resources, namely natural resources (e.g. fisheries, pastures and forests) as well as the immaterial ones such as knowledge.<br />
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The commons' management has been belittled for a long time. Indeed, since the biologist Garett Hardin published his famous article entitled “The tragedy of the commons” in Science in 1968, the negative arguments he gave against the commons have tarnished it. Hardin postulates that one pursues its own interest and as a consequence if a resource (e.g. a pasture) were to be exploited freely by anyone, it would rapidly be destroyed as everybody would try to take the best slice out of it. Hardin concludes : “Ruin is the destination toward which all men rush, each pursuing his own best interest in a society that believes in the freedom of the commons. Freedom in a commons brings ruin to all.”<br />
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However, Elinor Ostrom's (1933-2012) work, which led her to win the Nobel prize in economics in 2009, broadened the economists' mind on this particular topic.<br />
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<div class="petitSsTitre">The new commons</div><br />
Ostrom emphasizes the fact that Hardin “was actually discussing open access rather than managed commons” (Hess & Ostrom, 2006). Using this argument among others, she pointed out the weaknesses of Hardin's thinking : contrary to his theory, a lot of resources have been responsibly managed as communal goods over many centuries in Europe, before being privatized through the “enclosure” movement from the 15th to the 18th century.<br />
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Her study made her precise the commons' concept. Throughout many examples, she ended up characterizing it as a “jointly owned legal set of rights”. Besides, in contrast to Hardin who offered only two solutions (privatization or nationalization) to the supposed “tragedy of the commons”, she distinguished this form of property and management of resources from both traditional private (market, companies) and public (planning, State) economic approaches: each community managing a commons fixes its own rules by defining a governance frameworkl and some “bundles of rights”. The latter which specifies the access, withdrawal, management, exclusion and alienation rights are distributed to the different actors exploiting the resource in order to manage it jointly, and efficiently according to a defined hierarchy.<br />
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Ostrom insisted on the uniqueness of every case of commons she (or her students) studied. Nevertheless she succeeded in identifying a set of founding principles each sustainable commons fits.<br />
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<ul><br />
<li>“clearly defined boundaries should be in place</li> <br />
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<li>rules in use are well matched to local needs and conditions</li><br />
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<li>individuals affected by these rules can usually participate in modifying the rules</li> <br />
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<li>the right of community members to devise their own rules is respected by external authorities</li><br />
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<li>a system for self-monitoring members’ behavior has been established</li> <br />
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<li>a graduated system of sanctions is available</li> <br />
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<li>community members have access to low-cost conflict-resolution mechanisms"</li><br />
</ul><br />
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<h3>Conclusion</h3><br />
<br><br />
The huge energetic and environmental challenges every country will have to face in the following years call for institutional and organisational innovations capable of promoting the development of relevant technological innovations. The success of the open source movement in the software industry tends to support the idea that patents are not the sole way to support knowledge spreading and technological innovations in an economic sector. Building commons can be considered, to some extent, as a fruitful alternative to the patent inflation and its pernicious effects.<br />
<br><br />
The iGEM community, as a group sharing knowledge on synthetic biology, can be regarded as a case of commons. But, as Elinor Ostrom's work highlights, it is the responsibility of every commons to define its own founding principles, namely a governance framework and an appropriate distribution of “bundles of rights” associated with different roles in the organization of the commons. Even if some milestones have been set, much remains to be done in this respect.<br />
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Consequently, we propose the iGEM community to develop a genuine reflexion on this important matter in order to form a strong “synthetic biology commons” whose technological and economic success could be compared to the open source movement.<br />
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The results of the UBC team’s survey, to the analysis of which we have collaborated, confirm that most of the iGEM community members oppose the patentability of the BioBricks designed in the frame of iGEM. Promoting the intellectual as well as economic value of iGEM outcomes, without resorting to the traditional intellectual property regime, should thus interest most actors in the iGEM commons.<br />
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<h3>Bibliography</h3><br />
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<br><ul><br />
<li>Thorstein VEBLEN (1908). "On the Nature of Capital. I. The Productivity of Capital Goods", <em>The Quarterly Journal of Economics</em>, Vol. 22, No 4, pp. 517-542</li><br />
<br><br />
<li>Garrett HARDIN (1968). "The Tragedy of the Commons", <em>Science</em>, New Series, Vol. 162, No. 3859, pp. 1243-1248</li><br />
<br><br />
<li>Charlotte HESS and Elinor OSTROM (2006). "Introduction: An Overview of the Knowledge Commons", pp. 3-26. In Charlotte HESS and Elinor OSTROM (eds). <em>Understanding Knowledge as a Commons</em>, Cambridge (MA), The MIT Press</li><br />
<br><br />
<li>Claude HENRY and Joseph E. STIGLITZ (2010). "Intellectual Property, Dissemination of Innovation and Sustainable Development", <em>Global Policy</em>, Vol 1, No. 3, pp. 237-251.</li><br />
<br><br />
<li> Learn more about Joseph E. Stiglitz and Elinor Ostrom's Nobel prizes : <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2001/stiglitz.html"> click here</a> or <a href="http://www.nobelprize.org/nobel_prizes/economics/laureates/2009/ostrom.html"> here</a></li><br />
</ul><br />
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<h2>Part II: Collaboration with UBC : IP issues encountered by iGEM teams</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<h3>Introduction</h3><br />
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The <a href="https://2012.igem.org/Team:British_Columbia">University of British Colombia team</a> was also interested in intellectual property issues. They sent to each team a survey to understand what kind of IP issues are encountered by iGEM teams? Do IP issues hinder iGEM project? What level of IP knowledge have iGEMers? We were glad to see we were not alone on IP planet, so we contacted them to share our works. UBC has accepted to give us their survey results and in exchange we commented their poll and their FAQ, we also answered their survey.281 iGEMers had answered the survey when we treated it .<br />
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This collaboration allowed us to broaden our reflexion to iGEM teams' issues.<br />
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<div class="petitSsTitre">Teams and IP experience</div><br />
Firstly we studied past iGEMers experience with IP . In a graphic we summarize the answer to two questions:<br />
<ul><br />
<li>Do you have a past experience regarding the IP ?</li><br />
<li>Was your past experience in the context of a past iGEM project or outside of it ?</li><br />
</ul><br />
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<center><img src="https://static.igem.org/mediawiki/2012/b/b9/Graphpastexperience.png" width="70%"/></center><br />
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Only a few iGEM participants experienced dealing with IP issues. However, it would have been interesting to know their age in order to know if both data were bounded.<br />
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Besides, a patent experience seems to be often linked to a different project than the iGEM one, the reason probably being the special functioning of the iGEM contest in which most of the tools needed (such as the plasmid vectors) are provided without any intellectual property rights. Outside of iGEM, of course, such tools are obviously not freely supplied, which may results in a patent experience if the materials used are under a proprietary license. <br />
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Nonetheless, these answers do not reveal what these past experiences are. To test our hypothesis (iGEMers acquire IP experience outside of iGEM because patented materials are not used) we got interested in the nature of iGEMers IP experiences.<br />
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<div class="petitSsTitre">Nature of the IP experience</div><br />
In this study, those who had an IP experience had to explain what was its nature. The results are that most of them (46 %) had already used patented materials before. However, 15% renounced to actually use them, which shows how patent can impede research and innovation: getting a license may be dissuasive, especially in France where the Research budget is limited.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/0/0b/Naturepastexperience.png" width="70%"/></center><br />
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The next answers are related to the current iGEM projects. The poll shows clearly that patents have been a problem for 10 % of the participants, which is not that much. But still : they do have been a concern in some cases. The relatively low number could be explained by the aforementioned reasons about the providing of non-patented tools by the iGEM organization.<br />
<br><br />
It could also be explained by the redundant solutions offered by the living matter in general and the synthetic biology in particular. For instance, our team specifically picked a Dispersin gene that was not patented, though others were available (whose commercial use was protected).<br />
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<center><img src="https://static.igem.org/mediawiki/2012/b/b0/Negative_effect_of_patent.png" width="70%"/></center><br />
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Nevertheless to the question : “have other IP concerns (copyright, trademark) have negatively affected your current iGEM project ?” about 67% of the participants answered yes, which is considerably more, but as we have not even been concerned by such a problem, we find it hard to explain why this number is so high.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/b/b6/Copyright.png" width="70%"/></center><br />
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Our sole conclusion on this matter is that the IPR often does affect iGEM projects, even if in general the patents are not the limiting IP tool for them.<br />
<br><br />
<div class="petitSsTitre">May a project be built on patented materials?</div><br />
19% of the survey participants said that their team’s project used patented materials. This means that privatized knowledge and the iGEM contest are compatible in some way or another. However, the potential industrialization of these teams’ work will probably be restricted because the commercial use of something built on patented material is forbidden. This is a shame as one of the iGEM objectives is to generate projects that may have economically viable applications.<br />
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It is also surprising to note that 48% of the participants do not know whether their work is based on patented information or not, which shows a lack of information on their project material. Besides, it could stab them in the back if they were wishing to industrialize their ideas.<br />
Several questions were asked on the use of patented material. As it was discussed before, it is the most important IP issue in iGEM. Nonetheless, only 20% of the survey participants said that their team’s project used patented material.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/c/ce/Patented_teamproject.png" width="70%"/></center><br />
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Furthermore, two thirds of the iGEM team members believe that using patented work would not stand in the way of patenting their own one. This is quite surprising because obviously one’s research cannot be patented if based on protected materials. This statistic is very interesting as it shows how much most of the iGEM community’s actors do not know their legal rights concerning the IP.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/6/6c/Protection.png" width="70%"/></center><br />
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<div class="petitSsTitre">Opinions on the patentability of BioBricks</div><br />
Two questions were asked to the participants :<br />
<ul><br />
<li>Do you think BioBricks CAN be patented in your country ?</li><br />
<li>And do you think they SHOULD be patentable ?</li><br />
</ul><br />
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<center><img src="https://static.igem.org/mediawiki/2012/1/19/Biobrick_can_be_patented.png" width="70%"/></center><br />
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To the first question both opinions were equally represented. Nevertheless, it would have been interesting to know the country for each answer, even if the IP legislation is quite similar in most of the represented countries.<br />
<br><br />
However, to the question “Should BioBricks be patentable ?”, more than a half (56%) answered “no”. This is probably linked to the fact that most iGEM competitors already use them without paying a license, so that they completely oppose such an idea. It also points out that most iGEM team members support the iGEM knowledge policy, which makes us believe that they would be inclined to acknowledge iGEM as a commons and would favorably respond to the changes resulting from the common reflexion we proposed at the end of the first part of this human practice study.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/d/d3/Biobrickshouldbe.png" width="70%"/></center><br />
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<div class="petitSsTitre">Different ways to get to know the IP protection procedures</div><br />
The iGEM competitors who planned to get their work protected used different ways to approach applying for IP protections. Three tendencies come forth : they did so by talking inside their team (to the graduate student advisor, to another team member or / and to a faculty advisor), but also outside of it (to an industry expert, to a member of the university’s commercialization office or to a legal professional). The last tendency was to search on the Internet.<br />
<br><br />
These inclinations are more or less equally represented in this poll, so that it does not reveal many things. The only really interesting point which has to be put forward is that most iGEM competitors who want to apply for IP protections do make some research on it, which emphasizes the complexity of such procedures and the lack of knowledge they probably had.<br />
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<center><img src="https://static.igem.org/mediawiki/2012/2/21/Applying_IP.png" width="70%"/></center><br />
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<div class="petitSsTitre">Conclusion</div><br />
<br><br />
<br><br />
Our work with the University of British Colombia made us realize how often the intellectual property rights interfered with the different iGEM teams’ project. Nonetheless, we were disconcerted when we found out the patents were not the main problem. Indeed, their source was copyrights and trademarks .<br />
<br><br />
Besides, this survey deepened our main reflexion (q.v. part I) as it emphasized the problems due to the superimposition of the commons and the patent models. It also highlighted the global lack of knowledge of the iGEMers upon the intellectual property rights, which shows the necessity of informing about this particular topic. We sincerely hope we have helped to do so.<br />
</div><br />
</div><br />
<h2>Part III: Scientific popularization</h2><br />
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<h3>Introduction</h3><br />
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A lot of stereotypes and pieces of information circulate on sciences, so that we can sometimes find it hard to know where the truth is hidden. The public may sometimes look for a reliable source to make its own opinion. We are convinced that we have a role to play and have decided to involve ourselves in the researchers' night which is a european scientific popularization event.<br />
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<h3>More about the researchers'night</h3><br />
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Since 7 years, this event is taking place on a single September night in about 300 cities all over Europe. During the Researcher's night, everyone can come and explore science in engaging ways. The main goal of this night is to put researchers in touch with public concern, and explain what researchers are doing, how, and why it is important for everybody’s daily life.<br />
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</ul><br />
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This year, the theme is "Imagine the future": what future will emerge from research laboratories? How do the scientists imagine the future? Archeology, Physic, Phylososphy, Biosciences or Linguistic: researchers share their lab experiences and thoughts on the future.<br />
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For the first time, the Lyon-INSA iGEM team is taking part to this event. We will discuss how synthetic biology can help in the future, but also what the potential negative impacts of this new discipline are. We would like people not to think to corn only when they hear the word "GMOs". To do so we decided to focus on medical application of GMO's as insulin, growth hormone, artemisine,...<br />
We have to be opened to all possible questions and be prepared to face a very heterogeneous public.<br />
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<br><br />
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<em>This event will take place on Friday night, 28 september 2012 at CCO Villeurbanne.<br />
That is why this part is so short. The wiki is freezing in a few hours from and we will describe our adventures later if we get to go to Boston. If you want to read it we will be there!</em><br />
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<em> Special thanks to our faculty advisor O. Brette for his advice and for his working late to help us and to the UBC team for sharing its poll results with ours. </em><br />
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<h1>Video presentation of Biofilm Killer</h1><br />
<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtsdia?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtsdia_our-project-presentation-biofilm-killer-lyon-insa-igem_tech" target="_blank"><font color ="purple">Our project presentation Biofilm Killer Lyon...</font></a></center><br><br />
<div ><h1>Biofilm Killer machinery</h1></div><br />
<center><img src="https://static.igem.org/mediawiki/2012/thumb/8/86/Schemapageproject.png/800px-Schemapageproject.png" width="55%"/></center><br />
<div ><h1>Team project description</h1></div><br />
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Biofilms are responsible for billions of dollars in production losses and treatment costs in the industry every year. Biofilm-related problems are major concerns in the food industry where they can cause food spoilage or poisoning, in health industry because of pathogens' persistence and dispersal, or in the oil and water industry where they cause corrosion. Assuming that the environment is already over-saturated with harmful chemicals such as biocides, whose long term health effects remain to be elucidated, <b>there is a great need for novel solutions to reduce detrimental biofilm effects</b>.<br/><br />
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<b>To reduce the use of biocides</b>, the INSA-Lyon iGEM team aims to <b>engineer bacterial "torpedo" capable of infiltrating and destroying biofilms</b> formed on industrial equipments, pipes or reservoirs. Industrial surfaces will then be protected from further deleterious contamination by either a <b>surfactant coating</b>, or the <b>establishment of a protective biofilm</b>.<br/><br />
<br/><br />
<br />
<br />
<center><iframe frameborder="0" width="320" height="320" src="http://www.dailymotion.com/embed/video/xtrxw3?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtrxw3_bacillus-swimming-into-a-biofilm_tech" target="_blank"><font color ="purple">Bacillus swimming into a biofilm</font></a></center><br />
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Our experimental model consists of <b><i>Staphylococcus epidermidis</i> as the detrimental biofilm</b>, and <b><i>Bacillus subtilis</i> as the "Biofilm Killer" agent</b>. Three complementary modules will be constructed to arm our "Biofilm Killer" strain:<br/><br />
<br/><br />
<div class=indented2 style="margin:20px;"><br />
<li>The first step will be to fit <i>Bacillus subtilis</i> swimmers with both a <b>biocide and a scattering agent</b>. Irrigation of these active substances in the biofilm should be facilitated by the tunneling activity of these swimmers cells.</li><br />
<li>Then, to protect the surface which used to be the biofilm's substrate from next possible bacterial adhesion, we will engineer the <b>conditional production of surfactin</b>, a naturally toxic bio-surfactant produced by <i>B. subtilis</i> and displaying well-known antimicrobial properties.</li><br />
<li>Finally, the <b>conditional establishment of a competitive barrier flora</b>, constituted of <i>B. subtilis</i> in our model, will be achieved by inhibiting the main biofilm repressor <i>abrB</i> gene. </li><br/> <br />
</div><br />
<br />
<br />
<i>Bacillus</i> strains are non-pathogenic, and do not cause equipment degradation by corrosion: their settlement on surfaces represents a biocide-alternative strategy to prevent the formation of new dangerous biofilm. This project provides a potential economy, and environmental friendly solution for the control of unwanted biofilm. <br />
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<center><br />
<!-- DESCRIPTION DU PROJET --><br />
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<div><br />
<br/><br />
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<h3 align=center> <a href="https://2010.igem.org/Team:INSA-Lyon">2010</a> </h3><br />
INSA-Lyon -> Silver Medal <br/><br />
<br />
<br/><br />
<br />
<h3 align=center> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS">2011</a> </h3><br />
Lyon-INSA-ENS -> Gold Medal and <b> Best New BioBrick Device, Engineered </b> <br/><br />
<br />
<br/><br />
<br />
<br />
<h3 align=center> 2012 </h3><br />
Same same.... but different<br/><br />
<b>Lyon-INSA is back!!</b> <br/><br />
<br/><br />
<br />
You, <b>sponsor</b>, want to take part in this great adventure?? <br/><br />
Download our sponsorship proposal, contact us, and make a step toward an innovative future with us!!! <br/><br />
<br />
<br />
<br />
<u>Choose your language:</u><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/d/d9/Lyon-INSA_plaquette.pdf"><IMG SRC="https://static.igem.org/mediawiki/2012/2/23/Lyon-INSA_UKflag.png" height=3% width=3% TITLE="English version"></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/35/Lyon-INSA_plaquettefr.pdf"><IMG SRC="https://static.igem.org/mediawiki/2012/d/df/Lyon-INSA_franceflag.png" height=3% width=3% TITLE="French version"></a><br />
<a href="https://static.igem.org/mediawiki/2012/2/23/Lyon-INSA_plaquettege.pdf"><IMG SRC="https://static.igem.org/mediawiki/2012/4/46/Lyon-INSA_germanflag.png" height=3% width=3% TITLE="German version"></a><br />
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<br/><br/><br />
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<!-- FIN DE DESCRIPTION DU PROJET --><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/AchievementsTeam:Lyon-INSA/Achievements2012-09-27T03:29:26Z<p>Suxiaohui: </p>
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<br><br />
<h1>Achievements : What did we do ?</h1><br />
<div style="margin:40px;font-size:15px;"><br />
<br />
<br />
<div id="achievements"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%" style="margin-right: 25px;"/><br />
<div> We have <b>registered</b> our team.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have completed the judging form.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have set up a <b>team wiki</b>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have prepared a <b>poster</b> and a <b>presentation</b> for the Jamboree.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have submitted <a href="https://2012.igem.org/Team:Lyon-INSA/datapage"><b>6 documented parts</b></a> to the registry and showed that they work as expected. Among them, two <b>improvements</b> for existing <i>B. subtilis</i> shuttle vectors : fully characterized plasmids, with the full BioBrick prefix and suffix for the first time in the registry.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have explored <a href="https://2012.igem.org/Team:Lyon-INSA/safety"><b>safety implications</b></a> of the project as a whole.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have <b>collaborated</b> with <a href="https://2012.igem.org/Team:British_Columbia">British Columbia (UBC) team</a> to exchange knowledge about Intellectual Property. Moreover, we exchange with the other french teams through the <a href="http://sites.google.com/site/igemfrance/">iGEM France website</a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have incorporated <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><b>Human Practices</b></a> and <a href="https://2012.igem.org/Team:Lyon-INSA/modelling"><b>Modelling</b></a> into our design.<br></div><br />
<br> <br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We get our project closer to application by meeting with experts to consider <a href="https://2012.igem.org/Team:Lyon-INSA/Context"><b>industrialization</b></a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have presented our project and Synthetic Biology in many ways : on different <b>websites</b>, in <b>newspapers</b>, in <b>scientific events</b>, in order to inform a public as varied as possible.<br></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/AchievementsTeam:Lyon-INSA/Achievements2012-09-27T03:28:18Z<p>Suxiaohui: </p>
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<h1>Achievements : What did we do ?</h1><br />
<div style="margin:40px;font-size:15px;"><br />
<br />
<br />
<div id="achievements"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%" style="margin-right: 25px;"/><br />
<div> We have <b>registered</b> our team.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have completed the judging form.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have set up a <b>team wiki</b>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have prepared a <b>poster</b> and a <b>presentation</b> for the Jamboree.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have submitted six <a href="https://2012.igem.org/Team:Lyon-INSA/datapage"><b>documented parts</b></a> to the registry and showed that they work as expected. Among them, two <b>improvements</b> for existing <i>B. subtilis</i> shuttle vectors : fully characterized plasmids, with the full BioBrick prefix and suffix for the first time in the registry.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have explored <a href="https://2012.igem.org/Team:Lyon-INSA/safety"><b>safety implications</b></a> of the project as a whole.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have <b>collaborated</b> with <a href="https://2012.igem.org/Team:British_Columbia">British Columbia (UBC) team</a> to exchange knowledge about Intellectual Property. Moreover, we exchange with the other french teams through the <a href="http://sites.google.com/site/igemfrance/">iGEM France website</a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have incorporated <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><b>Human Practices</b></a> and <a href="https://2012.igem.org/Team:Lyon-INSA/modelling"><b>Modelling</b></a> into our design.<br></div><br />
<br> <br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We get our project closer to application by meeting with experts to consider <a href="https://2012.igem.org/Team:Lyon-INSA/Context"><b>industrialization</b></a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have presented our project and Synthetic Biology in many ways : on different <b>websites</b>, in <b>newspapers</b>, in <b>scientific events</b>, in order to inform a public as varied as possible.<br></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/AchievementsTeam:Lyon-INSA/Achievements2012-09-27T03:27:31Z<p>Suxiaohui: </p>
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<h1>Achievements : What did we do ?</h1><br />
<div style="margin:40px;font-size:15px;"><br />
<br />
<br />
<div id="achievements"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%" style="margin-right: 25px;"/><br />
<div> We have <b>registered</b> our team.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have completed the judging form.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have set up a <b>team wiki</b>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have prepared a <b>poster</b> and a <b>presentation</b> for the Jamboree.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have submitted six <a href="https://2012.igem.org/Team:Lyon-INSA/datapage"><b>documented parts</b></a> to the registry and showed that they work as expected. Among them, two <b>improvements</b> for existing <i>B. subtilis</i> shuttle vectors : fully characterized plasmids, with the full BioBrick prefix and suffix for the first time in the registry.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have explored <a href="https://2012.igem.org/Team:Lyon-INSA/safety"><b>safety implications</b></a> of the project as a whole.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have <b>collaborated</b> with <a href="https://2012.igem.org/Team:British_Columbia">British Columbia (UBC) team</a> to exchange knowledge about Intellectual Property. Moreover, we exchange with the other french teams through the <a href="https://sites.google.com/site/igemfrance/">iGEM France website</a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have incorporated <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><b>Human Practices</b></a> and <a href="https://2012.igem.org/Team:Lyon-INSA/modelling"><b>Modelling</b></a> into our design.<br></div><br />
<br> <br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We get our project closer to application by meeting with experts to consider <a href="https://2012.igem.org/Team:Lyon-INSA/Context"><b>industrialization</b></a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have presented our project and Synthetic Biology in many ways : on different <b>websites</b>, in <b>newspapers</b>, in <b>scientific events</b>, in order to inform a public as varied as possible.<br></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/AchievementsTeam:Lyon-INSA/Achievements2012-09-27T03:27:15Z<p>Suxiaohui: </p>
<hr />
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{{Lyon-INSA/datapage}}<br />
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<br><br />
<h1>Achievements : What did we do ?</h1><br />
<div style="margin:40px;font-size:15px;"><br />
<br />
<br />
<div id="achievements"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%" style="margin-right: 25px;"/><br />
<div> We have <b>registered</b> our team.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have completed the judging form.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have set up a <b>team wiki</b>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div> We have prepared a <b>poster</b> and a <b>presentation</b> for the Jamboree.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have submitted six <a href="https://2012.igem.org/Team:Lyon-INSA/datapage"><b>documented parts</b></a> to the registry and showed that they work as expected. Among them, two <b>improvements</b> for existing <i>B. subtilis</i> shuttle vectors : fully characterized plasmids, with the full BioBrick prefix and suffix for the first time in the registry.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have explored <a hrf="https://2012.igem.org/Team:Lyon-INSA/safety"><b>safety implications</b></a> of the project as a whole.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have <b>collaborated</b> with <a href="https://2012.igem.org/Team:British_Columbia">British Columbia (UBC) team</a> to exchange knowledge about Intellectual Property. Moreover, we exchange with the other french teams through the <a href="https://sites.google.com/site/igemfrance/">iGEM France website</a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have incorporated <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><b>Human Practices</b></a> and <a href="https://2012.igem.org/Team:Lyon-INSA/modelling"><b>Modelling</b></a> into our design.<br></div><br />
<br> <br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We get our project closer to application by meeting with experts to consider <a href="https://2012.igem.org/Team:Lyon-INSA/Context"><b>industrialization</b></a>.<br></div><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/9c/D%283%29.gif" width="3%"style="margin-right: 25px;"/><br />
<div>We have presented our project and Synthetic Biology in many ways : on different <b>websites</b>, in <b>newspapers</b>, in <b>scientific events</b>, in order to inform a public as varied as possible.<br></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
<br />
</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/notebookTeam:Lyon-INSA/notebook2012-09-27T03:19:50Z<p>Suxiaohui: </p>
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<div id="projectBar" class="menuBar"><br />
<div class="boutonMenu" onclick="window.location='menu';">Menu</div><br />
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<project><br />
<month name="July" size="31"><br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 2nd 2012</date><br />
<description>Liquid culture (5 mL LB medium) of NM 522 cells and overnight incubation under agitation at 37°C for later transformation.</description> <br />
</jour><br />
<jour nb="3"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 3rd 2012</date><br />
<description><p>Dilution of 100 µL saturated culture in 5 mL LB medium. </p><br /><br />
<p>Incubation time : 2 hours (until OD<sub>600</sub> = 0.3). </p><br /><br />
Transformation of the NM522 strain (this experiment was repeated 3 times). <br />
<ul><br />
<li>For the positive control the pSB1C3 plasmid was used;</li><br />
<li>For the transformation, the pBBA_I742123 was used (well A2, iGEM 2012 kit plate 5).</li><br />
</ul><br />
The transformed bacteria were selected on chloramphenicol (Cm) plates.<br />
</description><br />
</jour><br />
<jour nb="4"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 4th 2012</date><br />
<description><br />
Transformation analysis :<br/><br/><br />
<ul><br />
<li>Positive control : lots of colonies;</li><br />
<br />
<li>Negative control : one of the three negative controls was contaminated (the correspondent transformed colonies were not used). No colonies were observed on the two other negative control plates;</li><br />
<br />
<li>Test plate : between 1 and 8 were observed.</li></ul><br />
<br/><br />
4 liquid cultures (5mL LB medium + 50 µL chloramphenicol) and 4 streaked chloramphenicol plates were made using isolated colonies likely to contain transformed bacteria.<br/><br/><br />
<br />
The antibiotic resistance to Ampicillin, Tetracyclin, Chloramphenicol and Kanamycin of the following strains was tested : BS 168, BS 168 MCherry, BS 168 GFP, BS 168 Lysostaphin, <i>Staphylococcus epidermidis</i>, BS <i>abrB</i>.<br />
</description><br />
</jour> <br />
<jour nb="5"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 5th 2012</date><br />
<description><br />
Antibiotics resistance tests :<br/><br/><br />
<ul><br />
<li>Bs 168 : no resistance;</li><br />
<li>Bs 168 M cherry : no resistance;</li><br />
<li>Bs 168 GFP : no resistance;</li><br />
<li>Bs <i>abrB</i> : Cm resistant;</li><br />
<li>Bs 168 lysostaphin PWG100 : no resistance;</li><br />
<li><i>S. epidermidis</i> : Tet resistant.</li><br />
</ul><br />
<br/><br />
Extraction of 4 clones containing the pBBA_I742123 plasmid. Verification by gel electrophoresis (after <i>Eco</i>RI digestion)<br />
<br />
</description><br />
</jour><br />
<jour nb="6"><br />
<titre>For all purposes</titre><br />
<date> Friday, July 6th 2012</date><br />
<description><br />
<br />
Phone conference with Romain Briandet.<br/><br/><br />
<br />
Terminator was retrieved from plate 1 well 13D.<br/><br/><br />
Long meeting.<br />
<br />
</description><br />
</jour><br />
<jour nb="9"><br />
<titre>For all purposes</titre><br />
<date> Monday, July 9th 2012</date><br />
<description><br />
<ul><br />
<li>pBBa_I742123 was put in storage (under the reference pBK1).</li><br />
<li>A liquid culture of NM522/pBK1 transformed cells was made so we could extract more plasmid DNA (50 mL LB medium + 500 µL Chloramphenicol + 500 µL liquid culture of transformed bacteria ).</li><br />
<li>The 3 genes coding for lysostaphin, dispersin and <i>lacI</i> repressor in pUC57 Ampicillin resistant plasmids were delivered.</li><br />
<li>Transformation of NM522 strain with pUC57-Lysostaphin, pUC57-Dispersin and pUC57-<i>sfp</i>.</li><br />
<li>200 µL of each transformed strains were spread on LB + Ampicillin plates.</li><br />
<li>Liquid cultures of the following strains were made : Bs 168 in LB, Bs <i>abrB</i> in LB + Chloramphenicol, <i>S. epidermidis</i> in LB + Tetracyclin</li></ul><br />
<br />
</description><br />
</jour><br />
<jour nb="10"><br />
<titre>For all purposes</titre><br />
<date> Tuesday, July 10th 2012</date><br />
<description><br />
<ul><br />
<li> The following parts were put in storage :</li><br />
<ul><br />
<li>Lysostaphin in pUC57 Amp resistant (pBK2);</li><br />
<li>Dispersin in pUC57 Amp resistant (pBK3);</li><br />
<li>Surfactin part 2 (RBS-<i>lacI</i>-terminator) in pUC57 Amp resistant (pBK4).</li><br />
</ul><br />
<li> and the following strains (in LB broth supplemented with required antibiotic) :</li><br />
<ul> <br />
<li><i>S. epidermidis</i> = BK1 (Tet<sup>R</sup>);</li><br />
<li>Bs <i>abrB</i> = BK2 (Cm<sup>R</sup>);</li><br />
<li>Bs 168 = BK3.</li><br />
</ul><br />
<li>Ligation of Dispersin and Lysostaphin biobricks :</li><br />
<ul><br />
<li>digestion of pBK2 with the restriction enzymes <i>Eco</i>RI and <i>SpeI</i>;</li><br />
<li>digestion of pBK3 with the restriction enzymes <i>PstI</i> and <i>XbaI</i>;</li><br />
<li>digestion of pBK4 with the restriction enzymes <i>EcoRI</i> and <i>PstI</i>;</li><br />
<li>3A ligation of the digested parts;</li><br />
<li>electrophoresis control showed the expected fragment for lysostaphin and dispersin (2.1 kb). However, the digested backbone plasmid had 3 fragments instead of 2.</li></ul><br />
<li>Plasmid A2 extraction with a midiprep (pBK5) and digestion → 3 bands are observed : <strong>PROBLEM. Digestion is made again with E, P and E+P → There are 2 <i>PstI</i> sites !!! WRONG PLASMID</strong></li><br />
<br />
<li>Transformation of NM522 strain with the part BBa_K606061 Chloramphenicol resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_B0010 Ampicillin resistant.</li><br />
<li>Transformation of NM522 strain with the part BBa_K606040 Chloramphenicol resistant.</li><br />
<li>Design and order of the primers for the constitutive promoter (part BBa_K143012).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, July 11th 2012</date><br />
<description><br />
<ul><br />
<li>Extraction of part BBa_K606061 (RBS) Chloramphenicol resistant from transformed NM522 strain.</li><br />
<li>Extraction of part BBa_B0010 (terminator) Ampicillin resistant from transformed NM522 strain. <i>(NB : the clones were pink which means that the part contains a RFP gene)</i></li><br />
<li>Extraction of part BBa_K606040 (pLacI) from transformed NM522 strain.</li><br />
<li>Extraction of pUC57-Lysostaphin/pUC57-Dispersin/pUC57-<i>lacI</i> from transformed NM522 strains.</li><br />
<li>Transformation of NM522 strain with the part BBa_0010 Ampicillin resistant. The observed phenotype does not correspond to the description found in the registry (the color of the colonies is pink). We decided to make another transformation with the same part, only this time the 2011 kit was used.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<titre>For all purposes</titre><br />
<date> Thursday, July 12th 2012</date><br />
<description><br />
<ul><br />
<li>PCR product electrophoresis of Promoter PCR : the product is not the good one.</li><br />
<li>Reception and storage of the primers (for the amplification of the BBa_K143012 part).</li><br />
<li>The transformed NM522 strain with the part BBa_0010 Ampicillin resistant from the 2011 iGEM kit shows the same phenotype as the part found in the 2012 kit.</li><br />
<li>Extractions with a miniprep kit are made :<br />
<ul><br />
<li>4 clones containing the P<sub>lac</sub> promoter (1,2,3,4);</li><br />
<li>4 clones containing the <i>Bacillus subtilis</i> RBS (1,2,3,4);</li><br />
<li>3 clones containing theTerminator (1,2,3);</li><br />
<li>4 clones containing the gene for Dispersin;</li><br />
<li>4 clones containing the gene for Lysostaphin;</li><br />
<li>4 clones containing the gene for <i>lac</i>I.</li><br />
</ul><br />
Then a digestion is made on a 0.7% agarose gel.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<titre>Kill</titre><br />
<date> Friday, July 13th 2012</date><br />
<description><br />
<ul><br />
<li>PCR of the constitutive promoter (part BBa_K143012) → the PCR did not work so we ran a new PCR with a more diluted promoter solution.</li><br />
<li>The following strains are put in storage:<br />
<ul><br />
<li>NM522 containing <i>lacI</i>-pUC57;</li><br />
<li>NM522 containing RBS-pUC57;</li><br />
<li>NM522 containing dispersin-pUC57;</li><br />
</ul><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date> Monday, July 16th 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
<ul><br />
<li>PCR of constitutive promoter → it didn’t work. A new PCR is run with modifications of settings. The annealing temperature was modified from 50°C to 57°C, and 3 solutions with different concentration were tested, however the concentration did not affect the yield.</li><br />
<li>Purification of the PCR product.</li><br />
<li>Gel electrophoresis of lysostaphin.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation of B0015 terminator.</li><br />
<li>Strains BK13 (Bs arbB), BK10 (Bs 168 GFP) and BK11 (Bs 168 mCherry) are put in storage.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date> Tuesday, July 17th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Long morning meeting to discuss results and to write some posters in a frenzied psychopath way in order to remember our tasks.</li><br />
<li>Ligation of promoter-RBS-<i>gfp</i> in plasmid.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168: buffers preparation.</li><br />
<li>Failure of B0015 transformation.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Cloning of the constitutive promoter in front of the dispersin part (the gene coding for Dispersin) and RBS/<i>gfp</i> (of <i>E. coli</i>) using a 3A ligation followed by transformation of the ligation in the NM522 strain;</li><br />
<li>Extraction of pUC57-lysostaphin. Gel electrophoresis showed a shaded DNA → RNase addition in the extraction kit buffer was forgotten.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> in 5mL of TSB (conditions described in protocol “Tests on Staphylococcus aureus biofilms in 24 well plate” ).</li><br />
<li>A bacterial suspension of <i>S. epidermidis</i> with OD<sub>600</sub> close to 0.132 is prepared from a Petri dish containing <i>S. epidermidis</i>. A microtiter plate is inoculated with 2 mL of the suspension diluted 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="18"><br />
<date> Wednesday, July 18th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Extraction and digestion (E & P) of Bs 168 strain GFP’s plasmid. Gel electrophoresis showed absence of non digested plasmid → extraction failure.</li><br />
<li>Genomic DNA extraction of <i>B. subtilis</i> 168 (Kit Genomic DNA from tissue).</li><br />
<li>Transformation of pBK5 in <i>B. subtilis</i> <i>abrB</i> failed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation results of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was unsuccessful, so it was repeated, this time with both a positive control (strain 39) and a negative one with NM522 culture ;</li><br />
<li>Addition of RNase to the miniprep from pUC57-lysostaphin clones. Gel electrophoresis : there is still a smear. However, the bands are as expected.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
<ul><br />
<li>The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i>.</li><br />
<li>A microtiter plate is inoculated with 2mL of a suspension obtained from diluting the liquid culture 1:100 with TSB supplemented with different concentrations of glucose in order to test the best conditions for biofilm formation. The plate is incubated during 24 hours at 37ºC.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="19"><br />
<date> Thursday, July 19th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>TSS tests are also made to be sure that all TSS solutions that we have are ok because some transformations did not work.</li><br />
<li>A 50µg/mL erythromycin solution is made in order to test the strains’ resistance;</li><br />
<li>Transformation of <i>yfp</i>, <i>gfp</i> and <i>cfp</i> (from iGEM plates) in NM522;</li><br />
<li>B0015 transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The transformation of the 3A ligation ([dispersin+RBS/<i>gfp</i>+pSB1T3]) was successful, so 6 clones were tested on a plate containing LB broth supplemented with Tetracyclin.</li><br />
<li>The fluorescence test of the transformed bacteria containing [dispersin+RBS/<i>gfp</i>+pSB1T3] confirm that the promoter is functional.</li><br />
<li>Ligation of the promoter in a Cm resistant iGEM plasmid was made in order to send it to the registry.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests : Testing the potential of S. epidermidis to form biofilms</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can conclude that the concentration of glucose has no impact on the adherence of <i>S. epidermidis</i> and that the fact of cultivating the strain in broth or on solid medium has no impact on the biofilm's quality.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date> Friday, July 20th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation results : all TSS work (except maybe 1 and 2 with a smaller yield) and promoter+pSB1C3 OK → 6 clones are isolated on a LB+Cm plate.</li><br />
<li>Quantification and gel electrophoresis of <i>B. subtilis</i> genomic DNA.</li><br />
<li>Antibiotic testing of <i>B. thuringensis</i> 407 <i>gfp</i> strain and other strains in LB+Ery growth medium.</li><br />
<li>Failure of transforming pBK5 in <i>B. subtilis</i> 168 with the same protocol as previously. New task : find a new protocol.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with the part BBa_K606030 (pBK6) and promoter+dispersin.</li><br />
<li>Ligation Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection) and transformation.</li><br />
<li>Ligation Lysostaphin in pSB1C3 and transformation.</li><br />
<li>Isolation of 6 clones of the Terminator transformation.</li><br />
<li>Transformation results of fluorescent genes : ok, except <i>yfp</i>. </li><br />
<li>NM522 strain containing pUC57-lysostaphin is put in storage under the name of BK12.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date> Monday, July 23rd 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Selected clones (NM522+pBK6) are red, meaning that P<sub>veg</sub> promoter and RBS from <i>B. subtilis</i> are recognized by <i>E. coli</i>’s ribosome. 4 clones are streaked in LB+Cm.</li><br />
<li>LB+Ery and TSB+Tet Petri plates are made.</li><br />
<li><i>yfp</i> transformation was successful.</li><br />
<li>GFP and CFP plasmids’ extraction showed a failure in ligation.</li><br />
<li><i>B. subtilis</i> 168 is put in storage (BK14) in TSB medium.</li><br />
<li>Extraction of B0015 terminator. Gel electrophoresis showed 4 good clones.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results</li><br />
<ul><br />
<li>Promoter+Dispersin in pIG23 : nothing on the plate;</li><br />
<li>Lysostaphin, negative control contains bacteria so the plate is put in junk.</li><br />
</ul><br />
<li>New digestions, ligations,transformations:</li><br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57;</li><br />
<li>Dispersin in pSB1C3;</li><br />
<li>Lysostaphin in pSB1C3;</li><br />
<li>Promoter+Dispersin in pIG23 (from Cobalt Buster Lyon-INSA-ENS 2011 Team collection).</li><br />
</ul><br />
<li>Streaking of 4 clones of the transformed strain NM522/pBK6 on a Cm + LB plate. All of them formed red colonies which shows that the constitutive promoter and the RBS specific to the <i>Bacillus subtilis</i> strain are recognized by the RNA polymerase of <i>E. coli</i>.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR of xylR. Gel electrophoresis : the PCR didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date> Tuesday, July 24th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Plasmid extraction from Bacillus strain (BT407 GFP). We tested 2 methods in parallel. However, only the one using the extraction kit seems to work.</li><br />
<li>Extraction of <i>gfp</i>, <i>cfp</i>, <i>yfp</i> ; test → OK : put in storage.</li><br />
<li>Extraction of the pHT315 GFP plasmid of <i>B. thuringiensis</i> 407 GFP to build a shuttle vector <i>B. subtilis</i> ↔ <i>E. coli</i>. Transformation in NM522 strain and selection on LB+Ampicillin medium : ok.</li><br />
<li><i>S. epidermidis</i> is put in storage in TSB medium under the reference BK15.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results:<br />
<ul><br />
<li>Lysostaphin+Dispersin in pUC57 : the plate is covered of clones;</li><br />
<li>Promoter+Dispersin : nothing on the plate;</li><br />
<li>Dispersin in pSB1C3 : a lot of clones;</li><br />
<li>Lysostaphin in pSB1C3 : a lot of clones.</li><br />
</ul><br />
Selection of 4 clones of each lysostaphin+iGEM plasmid and dispersin+iGEM plasmid on Cm plate and in a liquid culture. Isolation of lysostaphin+dispersin (because there are too many clones!)</li><br />
<br />
<li>Extraction of the plasmidic DNA [Promoter in pSB1C3].</li><br />
<li>Extraction of pBK6 from the transformed strain.</li><br />
<li>Extraction of the [Promoter+RBS+<i>gfp</i>] in pBK23 (Tet R) by miniprep and verification by electrophoresis after digestion by <i>Eco</i>RI and <i>Pst</i>I : gel ok. Congrats (pBK12) → 122,6 ng/µL.</li><br />
<li>Extraction of the pBK6 plasmid (P<sub>veg</sub>+RBS+RFP in pSB1C3). The electrophoresis confirms the plasmid’s structure.</li><br />
<li>Liquid culture of Bs168 pWG100 overnight in order to do the first lysostaphin tests on plates.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New PCR of xylR with some modifications in the protocol : the PCR didn’t work.<br />
<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date> Wednesday, July 25th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Strains are put in storage :</li><br />
<ul><br />
<li><i>S. epidermidis</i> in TSB+Tet (BK17);</li><br />
<li><i>B. thuringensis</i> 407 GFP in LB+Ery (BK16).</li><br />
</ul><br />
<li>Minipreps to extract the plasmidic DNA of the clones NM522 + pHT315 GFP and verification by electrophoresis : plasmid ok (digested by E, X, S, P and not digested).</li><br />
<li>Transformation attempt of Bs 168 by pBK5 with a new procedure : Failure → blaming the plasmid which seems not to be the expected plasmid... Try again with the shuttle vector pHT315 (GFP or not).</li><br />
<li>The plasmid extraction protocol from <i>B. subtilis</i> was tested once again, with a few modifications. The results were unsatisfying. After electrophoresis, only genomic DNA was observed.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of the extracted plasmidic DNA of the clones transformed by [Promoter in pSB1C3] : none of the 4 clones is ok.</li><br />
<li>Selection of 4 clones transformed by [lysostaphin+dispersin] on LB+Amp plates and in liquid cultures.</li><br />
<li>Miniprep to extract [Lysostaphin+Cm] and [Dispersin+Cm] → Digestion.</li><br />
<li>3A ligation between : lysostaphin in pUC57 + Terminator in pSB1K3 + pSB1C3. Verification by electrophoresis : Lysostaphin, terminator and vector are ok but not the ligation (no DNA).</li><br />
<li>Lysostaphin tests (Bs 168 pWG100 supernatant) on antibiograms (diameter : 6mm) applied on LB+Tet plates having a biofilm already established and in development of <i>S. epidermidis.</i></li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
PCR on the <i>B. subtilis</i> 168 genome using universal primers of Phil Oger, and positive control with <i>Bulkhoderia cepacia</i> genome provided by Phil Oger. Goal : determine if the PCR problems come from the primers or the low concentration of genomic DNA of <i>B. subtilis</i> 168. <br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date> Thursday, July 26th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Transformation of NM522 strain with pHT304 and pHT315 (2 shuttle vectors for <i>B. subtilis</i> and <i>E. coli</i>).</li><br />
<li>gDNA extraction with 2 different protocols : the first for <i>Bacillus subtilis</i> 168 and the second for Gram- bacteria with some modifications. The first protocol gives a better yield.</li><br />
<li>PCR of rRNA 16s on B.s. 168 to test if PCR works in the bacteria without being lysed : it works.</li><br />
<li>NM522 + <i>gfp</i>, <i>cfp</i> and <i>yfp</i> in storage.</li><br />
<li>pHT315 GFP put in storage under the reference pBK18.</li><br />
<li>NM522 + pHT315 GFP strain put in storage under the reference BK21.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Other clones transformed with the Constitutive Promoter in pSB1C3 are selected in order to do other verification by extracting their plasmidic DNA (but they are not good again).</li><br />
<li>Extraction of the plasmidic DNA of the clones transformed with Lysostaphin+Dispersin in pBK2 and verification by electrophoresis gel. The clones are not right.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A assembly RBS-<i>gfp</i> (pBK7-pBK14) in pSB1K3 = L1. Transformation of L1 in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date> Friday, July 27th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
Extraction of transformed clones (NM522/pHT304 and NM522/pHT315). The electrophoresis showed that the plasmids had approximately 7kb and the restriction sites <i>Xba</i>I, <i>Eco</i>RI and <i>Pst</i>I as expected. However, there is a <i>Spe</i>I site which had not been mapped.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA : Lysostaphin in pSB1C3 clones, Promoter in pSB1C3 clones.</li><br />
<li>Plasmidic DNA verification by digestion and electrophoresis : Lysostaphin clones okay and Promoter clones are not okay.</li><br />
<li>New 3A ligation with Lysostaphin/Terminator/pSB1C3 and transformation in NM522 strain.</li><br />
<li>Results of the lysostaphin tests on plates : no effect (regular biofilms)</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>sfp</i> (surfactin part 1) and <i>abrB</i> put in storage : pBK16 and pBK17.</li><br />
<li>Failure of transformation of L1 in NM522.</li><br />
<li>3A assembly of RBS-<i>cfp</i> (pBK7-pBK13) in pSB1C3 = L2. (!! the part2 construction already has a terminator)</li><br />
<li>3A assembly of <i>sfp</i>-part2(<i>lacI</i>)-terminator (pBK4-pBK10) in pSB1C3 = IV.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>PCR of <i>xylR</i> from gDNA extracted yesterday. Test on gel : successful PCR !!</li><br />
<li>3A ligation of RBS (pBK7) and <i>xylR</i> (produced by PCR) in pSB1C3 ( ! the vector plasmid has the same resistance as the plasmid containing the RBS...).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date> Monday, July 30th 2012</date><br />
<titre>For all purposes</titre> <br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li><strong>It was also a fire day : a man’s hair and a lab bench on fire !!!! As the saying goes, ”everything comes in threes, if it's happened twice, it'll happen a third time ” (we’re still waiting for the third fire…).</strong></li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion test and gel of pBK2 (promoter + lysostaphin) by <i>Eco</i>RI and <i>Spe</i>I, pBK3 (dispersin + terminator) by <i>Xba</i>I and <i>Pst</i>I and pSB1C3 by <i>Eco</i>RI and <i>Pst</i>I.</li><br />
<li>We got 3 clones for the transformation of NM522 bacteria with the ligation’s product Lysostaphin+Terminator-pSB1C3 : the plasmidic DNA of these 3 clones is extracted and tested by electrophoresis gel → the 3 clones aren’t right.</li><br />
<li>Lysostaphin test on plate : this time, by trying with biofilm in formation less dense (OD<sub>600</sub> = 0.5, dilution the culture x100, 1000 and 10 000).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of L1, L2 and IV in NM522.</li><br />
<li>Transformation of <i>sfp</i> and <i>abrB</i> in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Purification of <i>xylR</i>, produced by PCR.<br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date> Tuesday, July 31st 2012</date><br />
<titre>Kill</titre> <br />
<description><br />
Results of the lysostaphin tests on plates : negative. There is no biofilm : too diluted but even though, the colonies do not seem to be affected by the presence of Bs 168 pWG100 supernatant → have the Bs 168 pWG100 lost their plasmid? (cultivated without selection pressure, that is without erythromycin).<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the previous transformation didn’t work, we made another transformation of NM522 strain with the <i>abrB</i> and <i><i>sfp</i></i> parts.</li><br />
<li>Results of the transformations of L1, L2 and IV : ok. Selection of some clones in order to do minipreps.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of RBS and <i>xylR</i> and transformation in NM522 strain.<br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="August" size="31"><br />
<jour nb="1"><br />
<titre>For all purposes</titre><br />
<date> Wednesday, August 1st 2012</date><br />
<description><br />
<br />
<p>The transformation protocol for <i>Bacillus</i> was tested using the pHT315 GFP plasmid extracted from the <i>B. thuringiensis</i> BT407GFP strain.</p><br />
<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>Lysostaphin+terminator : nothing;</li><br />
<li>Lysostaphin+dispersin : a lot of clones.</li><br />
</ul><br />
Cultures in LB broth of Lysostaphin+Dispersin are launched.</li><br />
<li>Digestion of Promoter clones and lysostaphin clones followed by an electrophoresis : lysostaphin clones are okay, promoter clones are not okay.</li><br />
<li>pUC57 with Dispersin put in storage : pBK3.</li><br />
<li>Lysostaphin test returns ! This time, we used more <i>S. epidermidis</i> (OD<sub>600</sub> = 0.75 diluted 1000 times) and a control for the stability of pWG100 plasmid (spreading of 200 µL on LB + Ery plates), after the liquid culture without selective pressure. Spreading on LB plates without Tetracycline (but addition of Tetracycline into the Bs 168 pWG100 supernatant).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The strain NM522/pBK6 was put in storage under the reference BK22.</li><br />
<li>Transformations of the NM522 strain with the ordered constructions (<i>sfp</i> and <i>abrB</i> in pUC57 AmpR). The transformation was unsuccessful, so we did it again.</li><br />
<li>Quantification of <i>sfp</i> and <i>abrB</i> constructions provided by Genecust using the Nanodrop.</li><br />
<li>Miniprep of the L1, L2 and IV ligations : it didn’t work.</li><br />
</ul><br />
</description> <br />
</jour><br />
<br />
<jour nb="2"><br />
<titre>For all purposes</titre><br />
<date> Thursday, August 2nd 2012</date><br />
<description><br />
<ul><br />
<li>The transformation of the <i>Bacillus</i> strain failed because of contaminated LB broth, so a new transformation was attempted.</li><br />
<li>pHT304 and pHT315 plasmids supernumerary site <i>Spe</i>I deletion attempt by digestion, filling-in, ligation and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Extraction of plasmidic DNA from the Lysostaphin + Dispersin clones in Cm iGEM plasmid (pSB1C3) and from the BK12 strain clones. Plasmidic DNA is checked by digestions.<br /> Lysostaphin+Dispersin clones digestion : clones not okay.</li><br />
<li>Ligations of :<br />
<ul><br />
<li>Constitutive promoter in Cm iGEM plasmid;</li><br />
<li>Dispersin in Cm iGEM plasmid.</li><br />
</ul><br />
Ligation were verified by electrophoresis.</li><br />
<li>Results of the Lysostaphin tests : some Bs 168 pWG100 contaminated the plate. However, we noticed a halo of 2-3 mm around the <i>Bacillus</i> colonies where <i>S. epidermidis</i> didn’t grow due to lysostaphin activity.</li><br />
<li>New Lysostaphin test on LB+Tet plate with a negative control (10 µL of Ampicillin).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 5 clones of the transformed NM522 strain with RBS-xylR and 2 clones of the NM522 transformed with the <i>abrB</i> construction. Given the fact that the NM522 strain used for the transformations was contaminated, the electrophoresis showed unexpected results.</li><br />
<li>New ligation of RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <I>sfp</i> and <i>abrB</i> in NM522 strain didn’t work.<br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date> Friday, August 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Purification of the NM522 strain (because of the problems on the negative control during the transformation) and resistance tests on the purified clones.</li><br />
<li>The results of <i>Bacillus</i> transformation are inconclusive : there are only a few clones on the plate. However, the negative control has a few colonies too. Despite this result, we decided to verify six clones and extract their DNA.</li><br />
<li>A lot of clones for the transformation of the pHT304 and pHT315 plasmids (without <i>Spe</i>I site) on LB+Amp plates → Purification of 12 clones each.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Velvet replication of Lysostaphin+Dispersin in pSB1C3 transformation plate on LB + Amp.</li><br />
<li>Antibiotic resistance checked for 5 Lysostaphin+Dispersin in iGEM plasmid clones (clones 1 to 5).</li><br />
<li>BK12 strain verification : spreading on LB + Amp plate.</li><br />
<li>Ligation of Promoter+Dispersin in Cm iGEM plasmid followed by transformation of the 3 ligations.</li><br />
<li>Ligation were verified by electrophoresis.</li><br />
<li>Transformation of Promoter+Dispersin ligation.</li><br />
<li>PCR to check the presence of the promoter in the Promoter in Cm iGEM plasmid clones.</li><br />
<li>Preliminary test of the liquid lysostaphin (10 µL of tetracycline to kill <i>Bacillus</i>, 500 µL of Bs 168 pWG100 supernatant, 500 µL of <i>S. epidermidis</i> with OD<sub>600</sub>=1.2 (the strains are cultivated overnight at 30°C). This test shows a possible effect of lysostaphin (decrease of OD<sub>600</sub> with the time, during 3h30). To be repeated with a negative control without Bs 168 pWG100.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation (3A protocol) of pBK7, pBK13 and iGEM backbone pSB1K3 (=RBS+<i>cfp</i>+pSB1K3).</li><br />
<li>Ligation (3A protocol) of pBK7, pBK14 and iGEM backbone pSB1K3 (=RBS+<i>gfp</i>+pSB1K3).</li><br />
<li>Because of the difficulties to transform the bacteria with the plasmids containing <i>sfp</i> and <i>abrB</i>, we decided to do an electrophoresis directly on the constructions sent by Genecust. Result : the genes were not inserted in a pUC57 plasmid, although they should have been cloned.</li><br />
<li>Ligation of <i>abrB</i> and <i>sfp</i> in pSB1K3 and transformation in NM522.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
New ligation between RBS and <i>xylR</i> in pSB1A3 and transformation in NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date> Saturday, August 4th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results :<br />
<ul><br />
<li>The negative control is ok;</li><br />
<li>There are clones on every transformation plates (Promoter in pSB1C3, Dispersin in pSB1C3, Promoter+Dispersin in pSB1C3).</li><br />
</ul><br />
8 clones for each transformation are chosen and spread on LB+Cm and LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformations of <i>abrB</i> and <i>sfp</i> are a success !! :D</li><br />
<li>Liquid cultures of <i>abrB</i> and <i>sfp</i> clones are launched to do plasmid extractions.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Sorting of the RBS + <i>xylR</i> clones to eliminate the clones containing two relegated plasmids.<br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date> Sunday, August 5th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid culture of 6 NM522 clones with pHT304 S and 6 clones pHT315 S.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Plasmid extractions from 3 clones NM522/<i>abrB</i> and 5 clones NM522/sfp. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of <i>sfp</i> construction (at 800 bp) and <i>abrB</i> construction (at 500 bp).<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Liquid cultures of RBS+<i>xylR</i> clones are launched to do plasmid extractions.<br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date> Monday, August 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Plasmid extraction from 6 clones Bs 168 transformed by pH315 GFP : the electrophoresis of the digested plasmids showed that the clones had the right plasmid. The <i>Bacillus</i> transformation also worked, but the yield must be improved.</li><br />
<li>Miniprep and digestions by <i>Spe</i>I of the plasmid extracted from the 6 clones of NM522 with pHT304 S and NM522 with pHT315 S : Failure ! The <i>Spe</i>I site is still in the plasmids :’( </li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Selection of clones growing on LB+Cm plates and not on LB+Amp plates :<br />
<ul><br />
<li>4 clones Dispersin in pSB1C3;</li><br />
<li>7 clones Promoter pSB1C3;</li><br />
<li>5 clones Promoter+Dispersin in pSB1C3.</li><br />
</ul><br />
Liquid cultures in LB+Cm are made with these clones. Then extraction of plasmidic DNA.</li><br />
<li>8 clones Lysostaphin + Dispersin in pSB1C3 are spread on LB+Cm and LB+Amp to test their resistance.</li><br />
<li>Transformation of pSB1C3 and pSB1T3 in order to obtain a clone from the PCR matrix (red clone).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Transformation of the following ligations, in NM522 strain :</li><br />
<ul><br />
<li>pBK7+pBK13+pSB1K3 (RBS+<i>cfp</i> in Kanamycin resistant backbone);</li><br />
<li>pBK7+pBK14+pSB1K3 (RBS+<i>gfp</i> in Kanamycin resistant backbone).</li><br />
</ul><br />
<li>PCR of RBS-<i>abrB</i> and P<sub>xyl</sub> and purification of these PCR products.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Purification of the <i>xylR</i> gene.</li><br />
<li>Miniprep of RBS-xylR and electrophoresis test → the ligation failed again...</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Tuesday, August 7th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>There is a little plasmid pHT304 S and pHT315 S post filling-in, new digestion by Spel enzyme to eliminate the plasmids which are not full on the SpeI site in order to increase the amount of plasmids without <i>Spe</i>I site.</li><br />
<li>Transformation in NM522 and spreading on LB+Amp plates.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>8 new clones Lysostaphin+Dispersin in pSB1C3 are screened on Ampicillin. Ampicillin sensitive clones are put in liquid culture.</li><br />
<li>Verification of plasmids extracted the previous day and electrophoresis : Dispersin in pSB1C3 (clones not okay), Promoter+Dispersin in pSB1C3 (Clones not okay).</li><br />
<li>The transformation of pSB1C3 and pSB1T3 didn’t work.</li><br />
<li>New Lysostaphin liquid test with 3 measurements and a negative control (Bs 168 supernatant instead of Bs 168 pWG 100). This time, the cultures grew over 36 hours at 37°C (efficiency of the lysostaphin in stationary phase ?) → the test is positive, lysostaphin has a right effect on the <i>S. epidermidis</i> cultures.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>3A ligation : [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + [psB1T3].</li><br />
<li>Transformation of the ligation [P<sub>xyl</sub>-RBS-<i>sfp</i>] + [RBS-<i>abrB</i>] + pSB1T3 in NM522 strain and spreading LB+Tet plates.</li><br />
<li>Results of the transformation by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : Failure! The negative control has a few colonies : the LB+Kan plate was contaminated and the used LB medium too! The transformation is done again...</li><br />
<li>Purification of P<sub>xyl</sub> produced by PCR.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Wednesday, August 8th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 by pHT304 S and pHT315 S : 0 clone and 1 clone... this only clone is tested but the test is negative : the <i>Spe</i>I site is always here at 311 bp. The cultures of the transformation are concentrated and spread on LB plate : 0 clone for pHT304 S and 18 for pHT315 S but none of the clones is good. We must do the filling-in again...</li><br />
<li>Transformation of the pSB1C3 + RFP and pSB1T3 + RFP plasmids extracts of the iGEM plates to use them as tool of cloning after extraction.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Plasmidic extraction of Lysostaphin+Dispersin clones, then digestion and electrophoresis : clones not okay.</li><br />
<li>New ligations : <br />
<ul><br />
<li>Promoter+Dispersin in pSB1C3;</li><br />
<li>Lysostaphin+Dispersin in pSB1C3.</li><br />
</ul><br />
Then transformation in NM522.</li><br />
<li>Plasmidic extraction of the BK12 strain : plasmid okay.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Results of the transformations :<br />
<ul><br />
<li>Transformations of NM522 by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : ok.</li><br />
<li>Transformation by <i>sfp</i>-<i>abrB</i>-pSB1T3 : the negative control isn’t good, the transformation is done again...</li><br />
</ul><br />
<li>Miniprep of other clones transformed with the plasmid RBS + xylR : the electrophoresis shows that the ligation still isn’t good…</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Thursday, August 9th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Extraction of NM522/pHT315S. We expected a plasmid without a SpeI site. However, the selected clone contained a plasmid identical to pHT315 (the digestion by <i>Spe</i>I and <i>Eco</i>RI enzymes gives a fragment at 1000 bp). We decided to screen more clones (8 out of 17).</li><br />
<li>The transformation pSB1C3 + RFP and pSB1T3 + RFP is a success ! (red clones)</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
Transformation results : <br />
<ul><br />
<li>Positive control : okay;</li><br />
<li>Negative control : okay;</li><br />
<li>[Lysostaphin + Dispersin] : 8 clones;</li><br />
<li>[Promoter + Dispersin] : more than 30 clones.</li><br />
</ul><br />
Clones are screened on LB + Amp and LB + Cm plates.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>The transformation of NM522 strain by <i>sfp</i>-<i>abrB</i> didn’t work again : although the positive control contains many clones (plasmid A2) and the negative control contains nothing, the real transformation contains nothing either ! We decided to do the <i>sfp</i>-<i>abrB</i> ligation again...</li><br />
<li>Plasmid extraction of 4 clones NM522 transformed by pBK7+pBK13+pSB1K3 and pBK7+pBK14+pSB1K3 : the electrophoresis of the digested plasmids showed that 3 out of 4 clones had the good fragment.</li><br />
<li><strong>PROBLEM : WE RAN OUT OF LIGASE!!</strong><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Friday, August 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Deletion of the <i>Spe</i>I site from pHT315 and pHT304 plasmids and filling-in using the <i>Pfu</i> polymerase. This time we used gel electrophoresis to verify the plasmids after each purification.</li><br />
<li>Extraction of the plasmid containing the RFP gene in the pSB1C3 iGEM backbone (midiprep).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>New Lysostaphin tests are planned (on plates) : in this purpose, a culture of 60 mL of Bs 168 pWG100 is inoculated and then incubated overnight at 28°C.</li><br />
<li>None of the clones Lysostaphin+Dispersin grew in LB + Amp so LB cultures were inoculated.</li><br />
<li>Out of 29 clones, 18 Promoter+Dispersin clones are Ampicillin sensitive, LB cultures are launched with these clones.</li><br />
<li>Plasmidic extractions of these clones : clones not okay.</li><br />
<li>PCR to obtain pSB1C3 : the electrophoresis showed that there was no DNA, so another trial was done, but it was unsuccessful.</li><br />
<li>The strain NM522 with Constitutive Promoter + pSB1C3 was put in storage under the reference BK27.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Ligation of the <i>sfp</i> and <i>abrB</i> parts in an iGEM backbone.</li><br />
<li>The NM522 strain with <i>abrB</i>-pSB1K3 was put in storage under the reference BK25.</li><br />
<li>The NM522 strain with <i>sfp</i>-pSB1K3 was put in storage under the reference BK26.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Saturday, August 11th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Gel electrophoresis showed that the elimination of the <i>Spe</i>I site was not successful.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Sunday, August 12th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Culture of 60 mL of Bs 168 pWG 100 at 28°C for the lysostaphin tests on plates.<br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Monday, August 13th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>We identified the reason why the deletion of the site <i>Spe</i>I from pHT315 and pHT304 plasmids and filling-in was not successful : the <i>Pfu</i> enzyme requires Mg<sup>2+</sup> and the buffer we used did not contain any, so we decided to give it another try, this time with the proper buffer.</li><br />
<li>The extracted plasmids containing the RFP gene in the pSB1C3 backbone were digested and verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lysostaphin tests : Flasks of 25mL of Bs 168 pWG 100 filtered supernatant were cultivated on Friday and frozen at -20°C on Sunday (2 flasks of each). The control flask is filled with 25mL of LB broth. These flasks are then frozen at -80°C and freeze-dried overnight.</li><br />
<li>The gel electrophoresis of the digested plasmid [Lysostaphin + Dispersin] in pSB1C3 doesn’t correspond to the expected fragments. Similarly, the second electrophoresis shows that the clones with [Constitutive promoter + Dispersin] in pSB1C3 do not have the right plasmid : the digested fragments do not have the right size.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligation [<i>sfp</i> + <i>abrB</i> + pSB1A3] in the NM522 strain.<br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Tuesday, August 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The gel electrophoresis of the digested plasmids pHT315 and pHT304 after the final purification shows promising results, so the two plasmids were transformed in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive Promoter (extracted by PCR) in the pSB1C3 plasmid containing the RFP gene (purified by midiprep) in order to obtain the Constitutive Promoter in the pSB1C3.</li><br />
<li>Lysostaphin tests on plates return again ! The freeze-dried products are put in five times less water than at first. Volumes up to 100 µL of concentrated supernatant are applied on antibiograms which are used for tests on the <i>S. epidermidis</i> biofilm.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformation of the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] was successful, so 4 clones were put in liquid culture in order to be tested.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Standard ligation between pBK7 (=RBS in pSB1C3) and xylR (obtained by PCR) with 1µL of insert for 5µL of vector. The ligation was transformed into the NM522 strain. <br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Wednesday, August 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the pBK20 (pHT315) and pBK19 (pHT304) plasmids was successful. 12 clones per plasmid were tested.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>The lysostaphin test didn’t work : Valérie did the test again by applying first the supernatant on the paper disks before putting them on the plates, and by using TSM medium instead of LB broth.</li><br />
<li>Transformation of the ligation [pSB1C3 + constitutive promoter] in NM 522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
None of the 4 clones tested had the expected fragments, so we screened 6 others.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by pBK7 and <i>xylR</i> : the negative control contains nothing, the positive control (transformed with pBK5) is full of bacteria and the plate with NM522 transformed by pBK7 + <i>xylR</i> contains a lot of clones too. 8 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Second standard ligation between pBK7 (=RBS in pSB1C3) and <i>xylR</i> (produced by PCR) with more insert (3µL of insert for 2µL of vector). Transformation into NM522 strains. </li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Thursday, August 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones having integrated the modified shuttle vector (after filling-in). 4 clones seem to have the right plasmid.</li><br />
<li>The electrophoresis after digestion with <I>Eco</i>RI and <i>Spe</i>I showed that the <i>Spe</i>I site was eliminated. 4 clones were chosen for further tests. However, the DNA concentration was too low so the enzymes did not digest the plasmids.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of the Constitutive promoter (P<sub>veg</sub>) produced by PCR in an iGEM plasmid.</li><br />
<li>Result of the lysostaphin tests : didn’t work again. The Bs 168 pWG100 supernatant doesn’t contain any lysostaphin : the lysostaphin is probably degraded in one way or another (sensitivity to defrosting ?).</li><br />
<li>Result of the transformation with [pSB1C3 + promoter] : a lot of red clones, some white clones (20 - 30). Isolation of 6 white clones and plasmid extractions by miniprep.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Miniprep of 4 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The electrophoresis did not turn out as expected for any of the 4 clones, so we decided to screen 6 others.</li><br />
<li>Ligation of P<sub>xyl</sub> produced by PCR in an iGEM plasmid.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Ligation of <i>xylR</i> produced by PCR in an iGEM plasmid (pSB1K3).</li><br />
<li>Results of the transformation of NM522 by [pBK7 + <i>xylR</i>] (for the second ligation) : the plate with bacteria transformed by [pBK7 + <i>xylR</i>] contains a lot of clones and the controls are standard. 14 clones are selected to do liquid culture and extract their DNA.</li><br />
<li>Plasmid extractions from the 8 clones transformed by [pBK7 + <i>xylR</i>]. The electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they have just the vector without <i>xylR</i>.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Friday, August 17th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The plasmids (pBK19 and pBK20) from 2 clones were re-extracted using columns. This time, the digestion was successful. The tests showed that there was no <i>SpeI</i> site. However, the pHT315 plasmid also had no XbaI site. We digested the two plasmids with the XbaI enzyme and this time we incubated the digestion during one hour, instead of 10 minutes. We also tested the enzyme on another plasmid which had a <i>XbaI</i> site. The digestion of the control plasmid was partial.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones with the transformed 3A ligation (pBK22, gene <i>abrB</i> and pSB1A3 backbone). The gel verification showed that there were 3 clones having the expected profile. The plasmid was put in storage under the reference pBK29.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Plasmid extractions from the 14 clones transformed by [pBK7 + <i>xylR</i>] (with the second ligation). As for the first transformation, the electrophoresis of the digested plasmids showed that the clones do not have the right plasmid : they only have the vector without <i>xylR</i>.<br />
</description><br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Monday, August 20th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>pBK19 with no SpeI site was put in storage under the reference pBK25.</li><br />
<li>pBK20 with no SpeI site was put in storage under the reference pBK26.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Launch of 8 [Promoter+Dispersin] cultures.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + P<sub>xyl</sub>] : the plate with bacteria transformed by [pSB1T3 + P<sub>xyl</sub>] contains few clones and the controls are standard. 3 clones are selected to do liquid cultures and extract their DNA : no plasmid.</li><br />
<li>Results of the transformation of NM522 by [pSB1T3 + RBS-<i>abrB</i>] : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of the transformation of NM522 by [pSB1K3 + <i>xylR</i>] : the plate with bacteria transformed by [pSB1K3 + <i>xylR</i>] contains a lot of clones and the controls are standard. 3 clones are selected to do liquid culture and extract their DNA : they have just the vector without <i>xylR</i>.</li.<br />
<li>A new PCR of <i>xylR</i> is made, in order to increase the stock.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="21"><br />
<date>Tuesday, August 21st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Meeting at 9 o’clock.</li><br />
<li>Midiprep of the modified pBK25 and pBK26 shuttle plasmids (without the SpeI site). The extracted DNA was verified by electrophoresis.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK23 (= Constitutive Promoter + RBS + Lysostaphin in pSB1C3) with pBK25 (the shuttle vector <i>E. coli</i> - <i>B. subtilis</i>). Different ligations are made with different proportions of insert and vector. Transformation in NM522 strain.</li><br />
<li>Digestion of Lysostaphin in pSB1C3 and Dispersin in pUC57.</li><br />
<li>Verification of 8 [promoter-dispersin] clones : clones happen to be nonstandard.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Extraction of the plasmid containing the ligation [<i>sfp</i>+<i>abrB</i>+pSB1A3] from a liquid culture of transformed bacteria.</li><br />
<li>Results of the second transformation of NM522 by pSB1T3 and RBS-<i>abrB</i> : no clones.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Two standard ligations are done :</li><br />
<ul> <br />
<li>pBK7-<i>xylR</i> (<i>xylR</i> cut in X and P sites);</li><br />
<li>pBK24-<i>xylR</i> (<i>xylR</i> cut in E and S sites).</li><br />
</ul><br />
<li>Transformation of pBK7-<i>xylR</i> into NM522 strain.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Wednesday, August 22nd 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Result of the transformation of NM522 with [Constitutive Promoter + RBS + Lysostaphin] in pBK25 : all the controls are right, and there are a lot of clones on the different plates with the transformed bacteria. 14 clones are selected to do liquid cultures and extract their DNA.</li.<br />
<li>New trial of cloning : Digestion, Ligation, transformation to construct :</li><br />
<ul><br />
<li>[Lysostaphin + Dispersin] in pSB1C3;</li><br />
<li>[Lysostaphin + Dispersin] in the shuttle vector (vector for <i>E. coli</i> and <i>B. subtilis,</i>).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 P<sub>xyl</sub> clones were tested, but none had integrated the plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Verification of 6 [<i>xylR</i>-pSB1K3] clones : they only have the vector.</li><br />
<li>Transformation of [pBK24-<i>xylR</i>] into NM522 strain.</li><br />
<li>Results of [pBK7-<i>xylR</i>] transformation : lots of clones and no clones in the negative control. 12 clones are put in liquid culture for extraction.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>B. subtilis</i> 168 in 5mL of LB broth.</li><br />
<li>Liquid culture of <i>B. subtilis</i> <i>abrB</i> in 5mL of LB broth.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Thursday, August 23rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Cloning of the iGEM linker into the shuttle vector (pBK25 and pBK26) and transformation in the NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformations results : All controls are okay.</li><br />
<li>Too many clones on [Lysostaphin + Dispersin] in shuttle vector transformation plate, so some clones are selected and spread and a new LB plate with the appropriate antibiotic.</li><br />
<li>12 liquid culture in LB are launched for the clones [Lysostaphin + Dispersin] in pSB1C3.</li><br />
<li>Miniprep of 14 clones transformed with the ligation product [Constitutive Promoter + RBS + Lysostaphin] in pBK25 and electrophoresis to analyze the extracted DNA : the transformation is successful for 3 clones =)</li><br />
<ul><br />
<li>The first clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (1);</li><br />
<li>The second clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (2);</li><br />
<li>The third clone [Constitutive Promoter + RBS + Lysostaphin] in pBK25 is put in storage under the reference pBK28 (3).</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of [P<sub>xyl</sub>+pSB1T3] in NM522.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Results of [pBK24-<i>xylR</i>] transformation : most of the clones are red, however a few of them are white, which is good. 7 white clones are put in liquid culture for extraction.</li><br />
<li>Extraction of pBK7-<i>xylR</i> from 12 clones, and then a gel electrophoresis is run: 2 clones seem interesting. We ran a second gel to verify these 2 clones, but it showed us that they were not good.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
A microtiter plate is inoculated with <i>B. subtilis</i> 168 and <i>B. subtilis</i> <i>abrB</i> in order to compare the adherence of each strain. (see Protocol 'Tests of Bacillus subtilis adherence in 24 wells plate').<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Friday, August 24th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The transformation of the cloned shuttle vectors was successful, so 12 clones are put in liquid cultures for further tests.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
3A ligation of pBK4 (pUC57 containing the <i>lacI</i> gene) and pBK29 (pSB1A3 containing the <i>sfp</i> and <i>abrB</i> genes) in pBK24 (pSB1C3 containing a RFP gene). The cloned fragment was transformed in the NM522 strain.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>After the bad results with <i>xylR</i>, we decided to cut pBK10 plasmid in <i>Sma</i>I site, and ligate with <i>xylR</i>, doing a blunt ligation. We also decided to ligate <i>xylR</i> with <i>xylR</i>, creating a big polymer, which will be like a “pre-ligation” molecule.</li><br />
<li>Extraction of pBK24-<i>xylR</i> from 7 clones. Gel electrophoresis is run → ONE CLONE IS GOOD!! Meaning that it’s the first time we manage to ligate <i>xylR</i> successfully.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plate prepared the day before is analyzed. There is a significant difference between the adherence potential of the two strains. <i>B. subtilis</i> <i>abrB</i> strain forms thin layer biofilms while <i>B. subtilis</i> 168 doesn't form any specific kind of biofilms.<br />
</description><br />
</jour><br />
<br />
<jour nb="25"><br />
<date>Saturday, August 25th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Miniprep of 12 clones selected from transformed bacteria with the cloned shuttle vectors; we ran out of <i>Spe</i>I enzyme, so we could not digest the extracted plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
6 white clones from the ligation pBK24-<i>xylR</i> are purified and put in liquid cultures.<br />
</description><br />
</jour><br />
<br />
<jour nb="26"><br />
<date>Sunday, August 26th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Extraction from 12 clones of lysostaphin and dispersin in pBK26 (shuttle vector) ligation and gel electrophoresis. The gel didn’t show any good clone.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of 6 clones (3A ligation: pBK24, pBK4, pBK29). The electrophoresis showed that none of the tested clones had integrated the right plasmid.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Extraction from the 6 white clones (containing <i>xylR</i>-pBK26). Gel electrophoresis is run, but it didn’t show any good clone. <br />
</description><br />
</jour><br />
<br />
<jour nb="27"><br />
<date>Monday, August 27th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of Bs 168 strain with pBK28 (1) plasmid which contains the [Constitutive Promoter + RBS + Lysostaphin] in the shuttle vector.</li><br />
<li>Midiprep : Extraction of P<sub>veg</sub>-dispersin-pSB1C3 of clone 22.</li><br />
<li>BK33 strain was put in storage.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Given the fact that the digested plasmids extracted from transformed NM522 <i>E. coli</i> with the 3A ligation (pBK24, pBK4, pBK29) had unexpected fragments (and impossible to explain), the ligation was done again, this time changing the vector/insert ratio (1/1 and 1/3, as opposed to the first trial with a ⅙ ratio ).</li><br />
<li>Liquid cultures of two potentially good clones of the NM522 strain transformed with a 3A ligation (pBK22(promoter P<sub>xyl</sub>, RBS and <i>sfp</i> gene), <i>abrB</i> gene and pSB1A3 backbone) were made for miniprep (the two plasmids were extracted using the classic ABC protocol, with extraction solutions that we made, so the yield and the purity of the extractions were poor; that’s why we decided to extract the plasmids again, this time using an extraction kit).</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
No transformation was done, because we ran out of LB broth. <br />
</description><br />
</jour><br />
<br />
<jour nb="28"><br />
<date>Tuesday, August 28th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Meeting at 1:30 pm.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Ligation of [Constitutive promoter + Dispersin] in the shuttle vector pBK25.</li><br />
<li>Results of the transformation of BS 168 strain with the pBK28 (1) in the shuttle vector : the negative control is full of bacteria → Is the Bs 168 strain resistant to erythromycin ? Or is the erythromycin concentration too low ? <br /><br />
We made another transformation and we spread the transformed bacteria on LB+Ery plates with different erythromycin concentration (from 5 µg/mL to 10 µg/mL).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The transformations with the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios ) was successful. 12 clones per transformation are put in liquid cultures and then streaked on LB+Cm plates and LB+Amp plates.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Transformation of <i>xylR</i>-pBK10 (blunt end ligation) in NM522.<br />
</description><br />
</jour><br />
<br />
<jour nb="29"><br />
<date>Wednesday, August 29th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation of the previous ligation [promoter-dispersin-pBK25] in <i>E. coli</i> NM522.</li><br />
<li>Results of the second transformation of Bs 168 strain with pBK28 (1) in the shuttle vector : the negative control is still full of bacteria even though the erythromycin concentration was higher than the previous trial (0,5 µg/mL the first time, 5 and 10 µg/mL the second time).</li><br />
<li>We made (LB+Ery) plates with different erythromycin concentrations in order to make different tests.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Only one of the 24 screened colonies concerning the two ligations (3A ligation of pBK24, pBK4, pBK29, with two different insert/vector ratios) did not turn up to be red (LB+Cm plate) and did not grow on a LB+Amp plate. The clone was used to inoculate a liquid culture for an extraction.</li><br />
<li>Miniprep of the two clones containing the <i>sfp</i> and <i>abrB</i> genes. The gel electrophoresis did not turn out as expected, probably because the bacteria used for inoculating the liquid cultures were contaminated or did not come from the same colony. We decided to transform the low purity plasmids into the NM522 strain.</li><br />
<li>Given the fact that we were not very optimistic concerning the 3A ligation of pBK24, pBK4 and pBK29, we decided to do two more trials, this time using two ligated genes (<i>sfp</i> and <i>abrB</i>) coming from clones other than the one containing pBK22. However, the miniprep of the bacteria which were supposed to contain the two plasmids was unsatisfying, so we decided to do the 3A ligation with the low purity plasmids, isolated with the classic protocol because we were running out of time and we were behind the schedule.</li><br />
<li>In parallel, we decided to do a standard ligation in order to transfer the <i>lacI</i> gene into a plasmid having an antibiotic resistance other than Ampicillin. This way, in case the 3A ligations fails, we would not be forced to do again a 3A ligation because the plasmids would have different antibiotic resistances.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Transformation of <i>xylR</i>-pBK24 in NM522. The transformation of <i>xylR</i>-pBK24 is done in order to see if <i>xylR</i> was successfully ligated or not. If the bacteria are red, then <i>xylR</i> was not ligated.</li><br />
<li>24 clones containing pBK10-<i>xylR</i> are patched in LB+Amp growth medium, and then incubated.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="30"><br />
<date>Thursday, August 30th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Transformation results : too many clones on the control digestion not ligated vector so the transformation can’t be used for the transformation [Lysostaphin+Dispersin] in pSB1C3.</li><br />
<li>12 clones are put in culture from the transformation of [Lysostaphin + Dispersin] in the shuttle vector treated by BamHI.</li><br />
<li>Preparation of tubes for sequencing, launch of miniprep and midiprep.</li><br />
<li>Standard ligation between the shuttle vector pBK26 (pHT315 modified) and the pBK23 plasmid (= Lysostaphin in pSB1C3). Transformation of the ligation product in NM522 strain.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>All 5 transformations were successful. White colonies are selected (pBK24 is a plasmid containing RFP in the pSB1C3 backbone, so we can exclude the colonies having integrated the religated backbone) and tested on LB+Cm and LB+Amp plates.</li><br />
<li>The miniprep of the one clone that had the expected phenotype (obtained after the transformation of the 3A ligation of pBK24, pBK4, pBK29) was digested, but the gel electrophoresis did not turn out as expected.</li><br />
<li>The miniprep confirmed that the ligation containing <i>lacI</i> was successful.</li><br />
</ul><br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>The patched plate is used as a master for replica-printing. The replica plate contains LB+Kan growth medium. Results : only one clone didn’t grow in the replica plate, meaning that it might be a clone containing <i>xylR</i>-pBK10 ligation. The clone is put in liquid culture overnight.</li><br />
<li>Another test is run to confirm that pBK34 plasmid (<i>xylR</i>-pBK24) doesn’t contain xylR : we digested the plasmid using NdeI restriction enzyme. Since <i>xylR</i> contains a <i>Nde</i>I restriction site (pBK24 doesn’t contain it), if pBK34 is opened by the enzyme, it means that it contains <i>xylR</i>. Result : pBK34 doesn’t contain xylem.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="31"><br />
<date>Friday, August 31st 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
We have finally received the enzymes, so we tested the minipreps containing the modified shuttle vectors (containing the iGEM linker). The results are promising : 4 plasmids are digested by <i>Spe</i>I, so the ligation was successful. The four plasmids are further tested for the other 3 iGEM sites. To make sure that the site SpeI is not the initial site that we eliminated by filling-in and that the plasmid used for transformation and ligation was not contaminated with the original plasmid, a double digestion was done (<i>Eco</i>RI and <i>Spe</i>I). There was no fragment at 1,000 bp, so the ligation was successful.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Verification of [Lysostaphin-Dispersin] in the shuttle vector (pBK26) clones : clones are not good.</li><br />
<li>Verification of the miniprep for sequencing ([Promoter + Dispersin] in pSB1C3) : DNA is okay.</li><br />
<li>Result of the transformation of NM522 strain with the Lysostaphin in pBK26 :</li><br />
<ul> <br />
<li>The negative control is ok;</li><br />
<li>The positive control is full of colonies;</li><br />
<li>The transformation is full of colonies : 24 clones are selected (on plates and in liquid culture) in order to extract and check their DNA. </li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the saturated cultures with transformed bacteria containing the ligation of the <i>sfp</i> and <i>abrB</i> genes. The electrophoresis confirmed that this time the bacteria contained the right plasmids.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
<ul><br />
<li>Extraction of the good clone from the replica-printing. Then a electrophoresis gel is run : the blunt end ligation was successful.</li><br />
<li>The plasmid is digested by <i>Eco</i>RI, <i>Xba</i>I, <i>Spe</i>I and <i>Pst</i>I to find out if the problem comes from a bad site sequence. Result : the <i>Eco</i>RI restriction site is not good, therefore is not recognized by <i>Eco</i>RI enzyme.</li><br />
</ul><br />
</description><br />
</jour><br />
</month><br />
<br />
<month name="September" size="30"><br />
<jour nb="1"><br />
<date>Saturday, September 1st 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Digestion of Lysostaphin in the shuttle vector and dispersin in pUC57 and verification of digestion. Then the vector is treated with CALF protein and a ligation is made using different of insert concentration. Then the ligation is transformed with commercial cells (STLBÉ) according to the manufacturer protocol and another trial is made with higher concentration.</li><br />
<li>Verification of midiprep : Midiprep of Lysostaphin in pSB1C3 is okay but not the one of Dispersin in pUC57.</li><br />
<li>Miniprep of 14 clones NM522 transformed with Lysostaphin in pBK26. The digestions and electrophoresis show that 7 clones are ok ! They are put in storage under the references pBK38 (1) to pBK38 (6).</li><br />
<li>Extraction of 14 clones transformed with Dispersin in pBK25. Gel electrophoresis showed no good clones.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>One of the 12 screened clones transformed with the ligation of pBK22 and pBK39 has the expected fragments. Unfortunately, the plasmid is mixed with another one. To separate the two plasmids we decided to transform 1 µL of the miniprep into <i>E. coli</i> NM522 strain.</li><br />
<li>6 other clones with the transformed 3A ligation (done on August 29th) are screened.</li><br />
<li>Standard ligation of the <i>abrB</i> gene and the plasmid containing <i>lacI</i> in pSB1C3 backbone (pBK37) and transformation. In parallel, another ligation is attempted : [pBK37 + pBK29].</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="2"><br />
<date>Sunday, September 2nd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Liquid cultures containing the modified shuttle vectors with the iGEM linker are inoculated and incubated overnight.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Shuttle vector containing lysostaphin is treated with an alkaline phosphatase, then a ligation with dispersin is realized, followed by transformation in <i>E. coli</i> NM522 strain.</li><br />
<li>Dispersin is ligated with pBK26 and then digested by BamHI before transformation. BamHI digestion allows not to transform the circularized vectors which doesn’t contain the insert.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Plasmids from the 6 clones containing the 3A ligation are extracted and digested. Gel electrophoresis showed that one of the clones has the expected fragments. However, it was mixed with another plasmid. In order to separate them, we decided to transform the NM522 strain with 1µL of the miniprep.</li><br />
<li>12 clones for each ligation (done the day before) are screened.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="3"><br />
<date>Monday, September 3rd 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Midiprep of the cloned shuttle vectors (containing the iGEM linker).</li><br />
<li>Miniprep of pBK26 shuttle vector is done in order to increase the stock.</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>No clones are obtained on the plate lysostaphin+dispersin when we followed the manufacturer protocol even on the positive control. But few colonies appeared on plates done with our own protocol.These clones are put in liquid cultures.</li><br />
<li>Tests are launched to verify manufacturer cells. And other tests are launched to verify that our plates contained the appropriate antibiotic.</li><br />
<li>A transformation is made with our cells of their positive control.</li><br />
<li>Transformations of Bs 168 :<br />
<ul><br />
<li>Trial n°1 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK25 (=pBK28).</li><br />
<li>Trial n°2 : negative control with H<sub>2</sub>O, positive control with pHT315 GFP (pBK18), transformation with Lysostaphin in pBK26 (=pBK38).</li><br />
</ul><br />
The bacteria are spread on LB+Ery plates ([Ery]=1 µg/mL and [Ery]=10 µg/mL).<br /><br />
The transformation of NM522 with ligation Dispersin in pBK26 was successful.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li><i>abrB</i> gene obtained by PCR was cloned in pSB1T3 backbone.</li><br />
<li>6 clones transformed with the mixture of two plasmids are screened.</li><br />
<li>Miniprep of 12 clones per transformed ligation : only one had the expected fragments (ligation of the <i>lacI</i> and <i>abrB</i> genes). The clone is put in storage under the reference pBK39.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="4"><br />
<date>Tuesday, September 4th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium and Ampicillin at different concentrations ranging from 0 to 1.3 mg/mL are inoculated with bacteria containing the shuttle vectors (pBK35 and pBK36).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Lots of clones are obtained with our cells.So we have tried a transformation of commercial cells with bigger quantities of DNA.</li><br />
<li>Miniprep of clones obtained with STLB2 are made. Clones are not okay. A new transformation with the same ligation mix is made but with NM522.</li><br />
<li>Results of the transformations of Bs 168 : not coherent. However, the problem seems to be identified : it is the erythromycin solution. Indeed, the erythromycin must be diluted in ethanol and not in water and must be kept refrigerated at -20°C.</li><br />
<li>Standard ligations between :<br />
<ul><br />
<li>P<sub>lac</sub> in pSB1C3 (pBK9) digested by <i>Spe</i>I and <i>Pst</i>I and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Pst</i>I;</li><br />
<li>P<sub>xyl</sub> (amplified by PCR) digested by <i>Spe</i>I and <i>Eco</i>RI and [RBS-<i>gfp</i>] in pSB1T3 digested by <i>Xba</i>I and <i>Eco</i>RI.</li><br />
</ul><br />
These two ligation products are transformed in NM522 strain.</li><br />
<li>Acrylamide gels and samples are prepared for SDS-PAGE.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis showed that 5 out of 6 clones screened after the transformation with the miniprep containing two plasmids had the expected fragments. However, we had doubts and we decided to search for restriction sites in the sequences that the plasmid was supposed to contain to make sure we had the right construction. We found out that a digestion with <i>Eco</i>RV would give 5 fragments of different sizes, enough to confirm the presence of the expected genes. Unfortunately, the electrophoresis was disappointing.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A PCR is run in order to get RBS-<i>xylR</i>. The PCR failed (not enough DNA-polymerase).<br />
</description><br />
<titre>Biofilm</titre><br />
<description><br />
<ul><br />
<li>One plate with <i>S. epidermidis</i> to evaluate the adhesion.</li><br />
<li>One plate with <i>S. epidermidis</i> + supernatant of different cultures (lysostaphin, dispersin...).</li><br />
<li>Same plate as the one above + crystal violet.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="5"><br />
<date>Wednesday, September 5th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Few clones are obtained with commercial cells, there are not very efficient. A new transformation is made with the same ligation mix but in NM522.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<ul><br />
<li>Standard ligation of pBK22 (<i>sfp</i> gene) and pBK39 (<i>abrB</i>-<i>lacI</i>) and transformation into the <i>E. coli</i> NM522 strain.</li><br />
<li>A SDS-PAGE gel is run to analyze lysostaphin (in BK32 and BK35 strains) and dispersin (BK29 strain).</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="6"><br />
<date>Thursday, September 6th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Ampicillin resistance tests gave some surprising results : even at 1.3 mg/mL there is bacterial growth. However, the higher Ampicillin concentration is, the lower OD<sub>600</sub> of the liquid cultures is. Ampicillin resistance test of the two shuttle vectors did not show any significant difference between the two strains (BK37 and BK38). The result is not surprising, given the fact that both plasmids (pBK36 and pBK35) derive from the shuttle vectors pHT315 and pHT304 which have the same origin replication as pUC19. However, the test must be done again, this time with at least two samples for each Ampicillin concentration in order to be able to estimate an error.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Clones obtained on lysostaphin-dispersin plates are put in liquid culture.</li><br />
<li>Transformation of Bs 168 : new trial ! To improve our protocol, we took periodic OD<sub>600</sub>600 measurements in order to stop the cell growth at the best stage (just between the exponential and the stationary phase). The Bs 168 strain is transformed by :<br />
<ul><br />
<li>pBK 25 (= modified shuttle vector pHT304);</li><br />
<li>pBK 26 (= modified shuttle vector pHT315);</li><br />
<li>pBK 28 (= Lysostaphin in the modified shuttle vector pHT304);</li><br />
<li>pBK 38 (= Lysostaphin in the modified shuttle vector pHT315);</li><br />
<li>Dispersin in pBK26.</li><br />
</ul><br />
We spread 200 µL of each transformed cells on LB + Erythromycin ([Ery] = 16 µg/mL) plates.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones transformed with the ligation pBK22 and pBK39 are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
2 new PCR are done to have <i>xylR</i>, but they failed. <br />
</description><br />
</jour><br />
<br />
<jour nb="7"><br />
<date>Friday, September 7th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>A miniprep of potential lysostaphin-dispersin clones is made : no clones are okay.</li><br />
<li>Result of the transformation of Bs 168 : </li><br />
<ul><br />
<li>All the negative control plates are clear : the erythromycin concentration is high enough to do a selection;</li><br />
<li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to extract and analyze their DNA;</li><br />
</ul><br />
<li>Transformation of Bs 168 : new trial by electroporation. As the last transformation, the Bs 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li><br />
<li>A new SDS-PAGE is made using supernatants and pellets as samples. The samples were not concentrated enough, so the gels didn’t show any of the expected bands.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (<i>sfp</i>, <i>abrB</i> and <i>lacI</i>) would be higher.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
A new RBS-<i>xylR</i> PCR is run, now with different concentrations of genomic DNA and a different annealing temperature. The PCR was successful.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="8"><br />
<date>Saturday, September 8th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
Miniprep of the clones Bs 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction for <i>E.coli</i> with addition of Lysozyme to the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel → Maybe the DNA isn’t concentrated enough ?<br /><br />
New clones are put in liquid cultures in order to make other minipreps. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.<br />
</description><br />
<titre>Stick</titre><br />
<description><br />
Ligation of P<sub>lac</sub>-RBS-<i>xylR</i> and then transformation in NM522.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Each of the 24 wells of two microtiter plates are inoculated with 2mL of a suspension obtained from diluting the liquid culture 100 times with TSB enriched with 1% glucose The plate is incubated during 24 hours at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="9"><br />
<date>Sunday, September 9th 2012</date><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>Miniprep of the clones Bs 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li><br />
<ul><br />
<li>Bs 168 clone transformed by pBK25 has the right plasmid !! =)</li><br />
<li>Bs 168 clone transformed by pBK28 has the right plasmid !! <strong>BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</strong></li><br />
<li>Bs 168 clones transformed by pBK26 and dispersin in pBK26 : nothing on the gel... </li><br />
</ul><br />
<li>Transformation of Bs 168 GFP with the five same plasmids as before. The transformed bacteria are spread on LB + Ery plates, with two different concentrations : [Ery]=10 µg/mL and [Ery]=15 µg/mL.</li><br />
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li><br />
<li>Thanks to the good results, we put in storage the transformed <i>Bacillus</i> under the reference :</li><br />
<ul><br />
<li>BK41 : Bs 168 + pBK25</li><br />
<li>BK42 : Bs 168 + pBK28</li><br />
<li>BK43 : Bs 168 + pBK26 (to confirm)</li><br />
<li>BK44 : Bs 168 + Dispersin in pBK26 (to confirm)</li><br />
</ul><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The supernatant from the two microtiter plates are decanted. The wells of a third microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the dispersin. The supernatants are diluted in order to test different concentrations of the supernatants. The wells of a fourth microtiter plate are filled with the supernatant of the liquid culture which is supposed to contain the lysostaphin.<br />
</description><br />
</jour><br />
<br />
<jour nb="10"><br />
<date>Monday, September 10th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>4 transformations with the same amount of plasmid are done (pBK19,pBK20, pBK35 and pBK36) in order to characterize the transformation efficiency of the shuttle vectors.</li><br />
<li>Ampicillin resistance test was repeated, this time with 3 tubes per Ampicillin concentration for each plasmid.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
We decided to give another try and do the ligation once again, this time using 5 time less vector and a ⅛ ratio vector/insert. Given the fact that we are running out of time, we did two ligations hoping to construct the same biobrick: pBK22+pBK39 and pBK29+pBK37. Both ligations were transformed into the <i>E. coli</i> NM522 strain.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li><i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic.</li><br />
<li>According to the results there is an effect of lysostaphin but we observed an effect due to the initial OD<sub>600</sub> of cultures that were different, so we don’t know if the results can be used. A new protocol is tried out, culture are diluted to have the same OD<sub>600</sub>.</li><br />
<li>New SDS-PAGE 12% gel is done with just the supernatants. The samples preparation didn’t go well and they were not enough concentrated. Therefore the gel didn’t show anything.</li><br />
</ul><br />
</description><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
<ul><br />
<li>Measurement of the NM522 + P<sub>lac</sub> kinetics (with Cm) during 12h in a 96 well plate with and without different IPTG concentration addition. (1 mM; 0.5mM and 0.1mM)</li><br />
<li>In the same 96 well plate NM522 + P<sub>xyl</sub> kinetics is also measured during 12h with and without different xylose concentration. (2%; 0.5% and 0.2%)</li><br />
<li>Results : kinetics decreases so there is a problem. But it can be explained by the fact that the promoters need to be activated by a sigma factor which is active in the exponential phase and 12h is not enough. So this experiment has to be done during 24h.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The microtiter plates prepared the day before are analyzed. Each well is stained with crystal violet and subsequently OD<sub>600</sub> is measured in order to quantify the adherence of the strain. We can see that the biofilm is not adherent enough and it is destroyed no matter what kind of supernatant is tested. Unfortunately, we cannot say what the effect of the dispersin or lysostaphin is using this kind of test.<br />
</description><br />
</jour><br />
<br />
<jour nb="11"><br />
<date>Tuesday, September 11th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+disp in pBK26 and Romain Briandet’s strains (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>Seeding a 96 well plate with <i>S. aureus</i> to produce a biofilm (plate 2).</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Design of the primers required to sequence the modified sequences of the shuttle vectors (after elimination of SpeI site by filling-in and cloning of the iGEM linker).</li><br />
<li>Ampicillin resistance test was a complete failure : the negative control (NM522 strain grown in LB broth supplemented with Ampicillin at the usual selective concentration), so the Ampicillin that was used lost it’s biological activity.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
12 clones per transformation are screened.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>E. coli</i> strains which might produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. <i>S. epidermidis</i> cultures are launched with tetracycline or without tetracycline. Presence or absence of antibiotic doesn’t change test results. The only effect is that cultures grow more slowly with antibiotic. According to the results there is an effect of lysostaphin but we observed an effect of dilution due to the protocol, so we don’t know if the results can be used. So a new protocol is worked out.</li><br />
<li>SDS-PAGE is done using pellets, to find a good concentration.</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
Liquid culture of <i>S. epidermidis</i> is incubated without any agitation at 37ºC.<br />
</description><br />
</jour><br />
<br />
<jour nb="12"><br />
<date>Wednesday, September 12th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>We remove the supernatant in the plate 2 made on Tuesday and added Bs168+dispersin in pBK26 and Bs168+pBK26 with different dilutions. Observation of the plate at the confocal laser scanning microscope after 5 hours of incubation. Exploitation of the pictures with Imaris software. Dispersin seems to have an effect on the biofilm.</li><br />
<li>Reading of the plate 1 with a confocal laser scanning microscope (after an overnight incubation). Exploitation of the pictures with Imaris software.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
The transformed plates with the modified and unmodified shuttle vectors do not show any significant difference, so we decided to make another essay, this time with 3 transformation for each plasmid.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately, the screened clones did not integrate the expected plasmid.<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture with the new protocol. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. There is a problem with the results : the control is not good.</li><br />
<li>Miniprep of one clone containing dispersin in pBK26 is done. Gel electrophoresis showed that the clone was good. The plasmid is then put in storage (pBK41).</li><br />
</ul><br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>5 test tubes containing a lamella are inoculated with the culture prepared the day (<i>S. epidermidis</i>) before diluted 1:100 with TSB supplemented with 1% glucose.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> dispersin but without the dispersin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the lysostaphin gene.</li><br />
<li>Liquid culture of <i>B. subtilis</i> containing the same plasmid as <i>B. subtilis</i> lysostaphin but without the lysostaphin gene.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="13"><br />
<date>Thursday, September 13th 2012</date><br />
<titre>Experiments realized in Massy</titre><br />
<description><br />
<ul><br />
<li>Seeding of 2 96 well plate with <i>S. aureus</i> to produce a biofilm. Addition of supernatant of Bs168+pBK25 or Bs168+pBK26 or Bs168+pBK28 or Bs168+dispersin in pBK26 and Romain Briandet's strain (Bs168+pWG200) with different dilutions (straight, 1/10, 1/100).(plate 1)</li><br />
<li>OD<sub>600</sub> measurements with filtered supernatants.</li><br />
<li>Mobility test of Bs168 on agar 0.25% plate.</li><br />
</ul><br />
</description><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>The transformed plates with the modified and unmodified shuttle vectors do not show any undeniable difference, so we decided to make another essay, this time with 3 transformation for each plasmid.</li><br />
<li>Ampicillin resistance test is repeated (3 samples for each Ampicillin concentration).</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Another 12 clones per transformation were screened (from the transformation made on 10th September).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in tanks to test dispersin effect on a <i>S. epidermidis</i> culture. Strains used for the test are <i>Bacillus subtilis</i> strains which may produce dispersin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture. Dispersin has no effect on <i>S. epidermidis</i> culture according to the results.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
<ul><br />
<li>One of the tubes and lamella prepared the day before containing <i>S. epidermidis</i> cultures is quantified using crystal violet staining and OD<sub>600</sub> is measured. <i>S. epidermidis</i> cells are quite adherent on glass surfaces.</li><br />
<li>The supernatant of the four other tubes are decanted and tubes are filled with the supernatants produced by the four liquid cultures of <i>B. subtilis</i> prepared the day before in order to test the effect of lysostaphin and dispersin.</li><br />
</ul><br />
</description><br />
</jour><br />
<br />
<jour nb="14"><br />
<date>Friday, September 14th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<ul><br />
<li>Transformation efficiency tests confirm our supposition : there is no significant difference between the transformation yields of the 4 plasmids (unmodified shuttle vectors, pBK19 and pBK20 and modified shuttle vectors, pBK35 and pBK36).<br /><br />
Moreover, OD<sub>600</sub> of the bacterial cultures in LB medium at various Ampicillin concentrations shows no significant difference between the BK37 and BK38 strains.</li><br />
<li>TG1 (<i>E. coli</i>) strain containing pJIM2241 shuttle vector is put in storage (BK47).</li><br />
</ul><br />
</description><br />
<titre>Kill</titre><br />
<description><br />
<ul><br />
<li>OD<sub>600s</sub> test in tanks to test lysostaphin effect on a <i>S. epidermidis</i> culture. The strains used for the test are <i>Bacillus subtilis</i> strains which may produce lysostaphin. Supernatant is filtered and applied on the <i>S. epidermidis</i> culture.</li><br />
<li>SDS-PAGE is done with some changes in the protocol. The gels showed a band that matches with the expected lysostaphin molecular weight.</li><br />
</ul><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Gel electrophoresis of the extracted plasmids shows that there are 5 possible plasmids containing the ligated insert (P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>-terminator). The five plasmids are digested by EcoRV and the electrophoresis confirms that, this time, the plasmids have the expected fragments.<br />
</description><br />
<titre>Physiological tests</titre><br />
<description><br />
The four tubes and lamella prepared the day before are quantified using crystal violet staining and OD<sub>600</sub>is measured. The same result is obtained : <i>S. epidermidis</i> biofilms are not adherent enough and biofilms are destroyed when the growth medium is changed.<br />
</description><br />
</jour><br />
<br />
<jour nb="15"><br />
<date>Saturday, September 15th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the 2 shuttle vectors pBK35 and pBK36.<br />
</description><br />
<br />
<titre>Kill</titre><br />
<description><br />
OD<sub>600</sub> test in 96 well plate to test impact of lysostaphin and dispersin on a culture of <i>S. epidermidis</i> (test during 12 hours).<br />
</description><br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
LB plates containing a 0.3% agarose gel are inoculated with 5 μL of a 5 hour liquid culture of <i>B. subtilis</i> containing the plasmids used in transformations.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of the ligations between the plasmid pBK42 containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] and the shuttle vectors pBK35 and pBK36 into the NM522 strain .<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="16"><br />
<date>Sunday, September 16th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Screening of 12 clones per <i>Bacillus</i> transformation.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Screening of 15 clones transformed with the ligation pBK42+pBK35 and pBK42+pBK36<br />
</description><br />
<br />
<titre>Physiological tests : Mobility of B. subtilis</titre><br />
<description><br />
The diameter of the disks formed by <i>B. subtilis</i> is measured. The fact of introducing the plasmids did not impact the swarming mobility of our bacteria.<br />
</description><br />
</jour><br />
<br />
<jour nb="17"><br />
<date>Monday, September 17th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Erythromycin at different concentrations was spread on LB plates with in order to test the Erythromycin resistance of the <i>B. Subtilis</i> strain transformed with the shuttle vectors.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Unfortunately the screened clones (containing the ligations pBK42+pBK35 and pBK42+pBK36) did not have the right plasmid. Another ligation of the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] is attempted, this time after gel extraction and purification of the digested pBK42 plasmid with EcoRI and PstI. This way, the backbone is eliminated and the chances of ligating the construction instead of the plasmid are higher.<br />
</description><br />
<br />
</jour><br />
<br />
<jour nb="18"><br />
<date>Tuesday, September 18th 2012</date><br />
titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing LB medium supplemented with Erythromycin at different concentrations are inoculated with the <i>B. subtilis</i> strains containing the shuttle vectors pBK35 and pBK36 in order to test the antibiotic resistance. For the same purpose LB + Ery plates are spotted with the same cultures at different dilution ratios.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
30 clones containing the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the shuttle vector are screened.<br />
</description><br />
<br />
<br />
</jour><br />
<br />
<jour nb="19"><br />
<date>Wednesday, September 19th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
The strains that were supposed to contain the shuttle vectors did not grow, so we suspect that the clones are in fact mutants.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Miniprep of the transformed clones with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>]. The gel electrophoresis shows that only two of them have the expected fragments.<br />
</description><br />
<br />
<br />
<br />
<br />
</jour><br />
<br />
<jour nb="20"><br />
<date>Thursday, September 20th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
<i>Bacillus subtilis</i> transformation with the shuttle vectors pBK35 and pBK36.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
Transformation of <i>B. subtilis</i> 168 and <i>B. subtilis abrB</i> strains with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] in the pBK35 shuttle vector. <br />
</description><br />
<br />
<br />
</jour><br />
<br />
<br />
<br />
<jour nb="21"><br />
<date>Friday, September 21st 2012</date><br />
<titre>Plac and Pxyl kinetics</titre><br />
<description><br />
A 7-hour-long kinetics is made with P<sub>lac</sub> and P<sub>xyl</sub> to be sure to have measurement in the exponential phase. Only one IPTG and xylose concentration is used.<br />
P<sub>lac</sub> = IPTG 0.5% and P<sub>xyl</sub> = xylose 0.5%.<br /><br />
Moreover, a 24-hour-long kinetics is also made in a 96 well plate. The conditions are the same as the one done for 12h except that bacteria are inoculated in a 1/100 dilution (instead of 1/50).<br />
</description><br />
<titre>Kill</titre><br />
<description><br />
SDS-PAGE is done in order to detect Dispersin in <i>B. subtilis</i> and <i>E. coli</i>'s supernatants. The gels were not conclusive, and were not good due to an ammonium persulfate problem.<br />
</description><br />
</jour><br />
<br />
<jour nb="22"><br />
<date>Saturday, September 22th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
12 clones transformed with the shuttle vectors are screened.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A new SDS-PAGE is done for dispersin. A migration problem persists, due to ammonium persulfate. <br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
24 clones transformed with the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>abrB</i>-<i>lacI</i>] are screened.<br />
</description><br />
</jour><br />
<br />
<jour nb="23"><br />
<date>Sunday, September 23th 2012</date><br />
<titre>For all purposes</titre><br />
<description><br />
Multiple tubes containing 2 mL liquid LB medium are supplemented with Erythromycin at various concentrations in order to test the antibiotic resistance of the two shuttle vectors. Also, LB+Ery plates are spotted with liquid cultures with the <i>B. subtilis </i> strains containing the shuttle vectors.<br />
</description><br />
<br />
<br />
<titre>Kill</titre><br />
<description><br />
A last SDS-PAGE is run. This time the problems were successfully solved. But the gels were not conclusive due to a low dispersin concentration.<br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
6 of the 24 clones are put in liquid culture for further testing in LB medium supplemented with xylose at a concentration of 2%.<br />
</description><br />
</jour><br />
<br />
<jour nb="24"><br />
<date>Monday, September 24th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
Both strains grew in all tubes. However, there is a difference concerning OD<sub>600</sub> between the two strains. The test is repeated (with antibiotic concentrations ranging from 0 to 1.5 mg/mL) in order to make sure there is a notable difference between the two strains. </li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
<li> In order to characterize the construction [P<sub>xyl</sub>-<i>sfp</i>-<i>arbB</i>-<i>lacI</i>] two tests are made. To confirm the presence of <i>sfp</i> gene, we made emulsions with the filtered supernatant of the transformed strains and sunflower oil. Concerning <i>abrB</i> gene, a biofilm formation test was made in a 24-well microplate in order to compare the transformed strain to the wild-type strain. <br />
</li><br />
</description><br />
</jour><br />
<jour nb="25"><br />
<date>Tuesday, September 25th 2012</date><br />
<br />
<titre>For all purposes</titre><br />
<description><br />
The results of the antibiotic resistance in liquid culture show that the <i>B. subtilis</i> strain containing pBK35 is less resistant than the one containing pBK36.</li><br />
</description><br />
<titre>Surfactant</titre><br />
<description><br />
The emulsion test and the biofilm formation test confirm the presence of <i>sfp</i> and <i>abrB</i> genes; <br />
</description><br />
</jour><br />
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<h1>Our collections</h1><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/safetyTeam:Lyon-INSA/safety2012-09-27T03:10:25Z<p>Suxiaohui: </p>
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<h1>Safety</h1><br />
<div class="contenuTexte introduction" style="margin:20px"><br />
We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified <i>Bacillus subtilis</i> strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked also with an <i>Escherichia coli</i> strain and as a biofilm model with <i>Staphylococcus epidermidis</i>. <br />
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<h2>Researcher/Public/Environmental Safety</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their toxicity for example. For these reasons: the access of the laboratory was limited to those involved in the project and all work benches were cleaned every day and the whole lab cleaned every week.<br />
<br/> <br/><br />
CLEAN AREAS TO ENCOURAGE GOOD PRACTICES<br />
<br/> <br/><br />
Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider the fact of having a proper training in safety and security at the beginning of our experimental work prepared us to be more organised, responsible for our actions and respectful for those of others. To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.<br />
<br/><br/><br />
<strong>Global hazards and risks :</strong> Electricity and gas: we have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. Personal protective equipement, including appropriate labcoats, gloves and safety glasses were worn for lab experiments. No one was allowed to work alone in the laboratory. In addition, the french system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life saving diploma and are also trained for firefighting.<br />
<br/><br/><br />
<strong>Main chemical hazards and risks :</strong> Most of the reagents used are irritant, toxic and can be potential carcinogens (agarose, polyacrylamid, methanol, Ethidium bromide…)<br />
<br/><br/><br />
To minimize the impact of their use, they are manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials are clearly identified/labeled to avoid inappropriate mix between two non compatible solvents. All reagents are eliminated in the appropriate waste recovered barrels.<br />
<br/><br/><br />
We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates whithin the double strand DNA helix. Many biological processes can thus be affected such as transcription and replication. To avoid the dissemination of EtBr in the lab, it is stored and used in the same room where the electrophoresis gels are revealed. EtBr is NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. This room is locked by a key. The access to this room is forbidden to any person who does not carry a lab coat. Dedicated gloves are used for EtBr manipulation and must not leave the room. They are discarded in a specific trash barrel for genotoxines contaminated material to gather with stained gels. EtBr-contaminated material is further processed and decontaminated by an external service.<br />
<br/><br/><br />
Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.<br />
<br/><br/><br />
<strong>Main biological hazards and risks:</strong> All <i>Bacillus subtilis</i>, <i>Echerichia coli</i> and <i>Staphylococcus epidermidis</i> strains we used have a biosafety level of 1, which means they are not known to cause disease and have minimal environmental hazards.<br />
<br/><br/><br />
At this level the precautions concerning the lab-material are minimal: wearing gloves and face protection when needed. Any contaminated material is discarded in a trash can to be autoclaved within 2 days. The decontamination procedure is similar to any other applied to frequently encountered virus or bacteria (ex: washing hands with antibacterial soap, disinfecting any surface in the laboratory that has been exposed). Our work benches are decontaminated with ethanol 70% after each manipulation. And contaminated media and materials are autoclaved at least every 2 days to avoid any release in environment.<br />
<br/><br/><br />
All the students must have their vaccinations up to date. In case a student harbors an injury, it must be covered so it is not exposed to the bacteria. All rooms are equipped with with a first aid kit.<br />
In the building where the labwork was performed, no laboratory is manipulating pathogenic microorganisms, which limits the risks to students, and the recombination possibility between a pathogenic bacteria and the lab strains.<br />
<br/><br/><br />
For better protection the following basic safety rules applied for all students during lab work:<br/><br />
<ul><br />
<li> Be aware of the risks and hazards involved in any experiment</li><br />
<br />
<li>Know where the fire extinguisher, safety shower and general electrical circuit breaker are located in each room</li><br />
<br />
<li>No food or drink in the laboratory</li><br />
<br />
<li>Wear a buttoned up lab coat</li><br />
<br />
<li> Long hair need to be tied back</li><br />
<br />
<li>Contact lenses are forbidden except if people wear a face protection or safety glasses</li><br />
<br />
<li>Spillage needs to be cleaned up immediately</li><br />
<br />
<li>Lab benches are cleaned after each experiment</li><br />
<br />
<li>Technical and safety files are available near all the lab equipments</li><br />
<br />
<li>No chemical touching, sniffing or tasting</li><br />
<br />
<li>No mouth-pipeting</li><br />
<br />
<li>No returning unused chemicals to their containers</li><br />
<br />
<li>No hazardous material disposal down the drain</li><br />
</ul><br />
<br/><br />
This specific localization in a public research laboratory building allows us to benefit from researcher and student law safety (or under application in France) which are mandatory in each lab in France. For example, we have specific places for each kind of experimentation:<br />
<br/><br/><br />
Rooms dedicated for:<br />
<br/><br />
<ul><br />
<li>Baths</li><br />
<br />
<li> Electrophoresis migration</li><br />
<br />
<li>Incubators</li><br />
<br />
<li>Freezer and refrigerator</li><br />
<br />
<li>Autoclave</li><br/><br />
</ul><br />
We use our personal autoclave in the lab to decontaminate and sterilize our materials.<br />
</div> <br />
</div><br />
<br />
<h2>BioBrick parts safety issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
A GMO may contain harmful parts and be dangerous for the environment. The potential danger of a GMO is also due to its potential interactions with the environment. When an iGEM project is conceived, the team has to think about the possible interactions of their parts with the environment and design tests to document the effects of their parts on the environment. Synthetic biology is a science which worries people, so we have to provide them the insurance of the complete safety of our experiments.<br/><br />
<br/><br />
<strong>Our molecules : Lysostaphin, Dispersin, Surfactin</strong><br />
<br/><br/><br />
None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Lysostaphin, an endopeptidase specific to the cell wall peptidoglycan of <i>Staphylococcus</i>, is an extremely potent antistaphylococcal agent. Lysostaphin has been used in animals and topically in human against certain infections. Concerning the safety precautions it is indicated to not breathe gas/fumes/vapor/spray, to avoid contact with skin and eyes.">lysostaphin</span>, <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Surfactin is a lipopeptide antibiotic and is used as a surfactant to mediate flux of mono- and divalent cations, such as calcium, across lipid bilayer membranes. It is a powerful biosurfactant which can cause lysis of erythrocytes and bacteria. It is also known as a clotting inhibitor. However, considering the concentrations in which it is produced by bacterial cultures, the risk is minimal. In addition, the “Biofilm Killer” GMO will produce the molecule only locally, further reducing the risks.">surfactin</span> and <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|DispersinB is commercialized by Kane Biotech. It should be used in conjunction with an antibiotic or an anti-microbial, such as silver. Last year, Kane Biotech provided a brief document on DispersinB wound spray to the FDA, in preparation for the submission of an Investigational New Drug Application. They have started clinical trials in order to test the effect of this molecule on infected people. To date, there is no evidence for a negative effect of this molecule on human health.">dispersin</span>. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here. <br />
<br/><br />
<br/><br />
<strong>Our chassis</strong><br />
<br/><br/><br />
In our Biofilm Killer project, we aim to construct a bacterium able to destroy biofilms and to synthesize biocide molecules or a biosurfactant in order to develop a novel method to remove biofilms and clean surfaces in several industrial processes. So, we chose a strain that would have a minimum impact on the process itself, the quality of the product and biological treatment that could exists on site (waste water treatment plant).<br />
<br/><br/><br />
Our genetic constructions have been transferred into a strain of <i>Bacillus subtilis</i>. This species are well known by researchers but also by many industries such as the food industry. <i>Bacillus subtilis</i> is also widely used in the field as a biocontrol agent against plant pathogens.<br />
<br/><br/><br />
It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA)<br />
<br/><br/><br />
<div style="margin:30px;font-style:italic;">"The <i>Bacillus subtilis</i> species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1]</div><br />
<br/><br/><br />
But <i>Bacillus subtilis</i> is also known to produce endospores that are resistant to different treatments (heat, UV irradiation…). A possible impact on human health cannot be excluded. Indeed, it has been shown that the ingestion of endospores of non-modified <i>B. subtilis</i> does not prevent their germination into the gastrointestinal tract (GIT) [4]. The bacteria are even capable of growth in the lower part of the GIT. But, in this particular case, the consequence seems to be beneficial on health. The presence of <i>B. subtilis</i> in the GIT seems to be linked to the development of the gut-associated lymphoid tissue (GALT). This is one of the reason <i>B. subtilis</i> can be used as a probiotic.<br />
<br/><br/><br />
When the environment becomes unadapted to its growth, the bacterium induces the formation of an endospore in its intracellular compartment [2]. The core of the endospore contains the bacterial chromosome. The core is protected by a double layer membrane, called the forespore. This membrane is itself protected by the coat, composed by multilayered proteins. In <i>B. subtilis</i>, the Cot family proteins is widely involved in the formation of the coat. The resistance of the spore is dependant on the state of hydratation of the core. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism. In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.<br />
<br/><br/><br />
Sporulation can be hazardous to the environment and humans because spores are resistant to most cleaning procedure and thus can survive for a very long time, even in extreme conditions [3]. Spores can be dispersed in the environment and turn into germination process leading to the contamination of new areas previously devoid of this micro-organism.<br />
<br/><br/><br />
In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.<br />
<br/><br/><br />
The high spore resistance to adverse conditions imply that avoiding the induction of sporulation is one task that we need to consider in our project if we use a sporulant <i>B. subtilis</i>.<br />
<br/><br/><br />
If the cells were released in the environment after the germination of spores, we estimate the chance of survival to be minimal, because strain 168 has been modified and habituated to laboratory conditions. Even so, as mentioned below, <i>B. subtilis</i> is not a pathogenic bacterium.<br />
<br/><br/><br />
<strong>For all these reasons, and in the aim of an industrial application, we propose to introduce our genetic constructions in an NON-sporulant <i>B. subtilis</i> strain, such as QB1133.</strong><br />
<br/><br/><br />
The use of a non sporulant <i>B. subtilis</i> to host our genetic construction would not significantly impact neither the environment nor the human health. The odds of the bacteria surviving to the low pH of the stomach, and the very hard life conditions (anaerobia, pH, competition with the intestinal flora) are very low in the vegetative state.<br />
<br/><br/><br />
In addition to the use of non sporulant mutant of <i>B. subtilis</i>, we could enhance safety by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"<br />
<br/><br/><br/><br />
<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br/><br />
1 European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.<br />
<br/><br />
2 Morphogenesis of <i>Bacillus</i> Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)<br />
<br/><br />
3 Role of the Spore Coat Layers in <i>Bacillus subtilis</i> Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626<br />
<br/><br />
4 The Intestinal Life Cycle of <i>Bacillus subtilis</i> and Close Relatives, Duc, Tran T. Hoa, Claudia R. Serra, Adriano O. Henriques Nguyen K. M. Tam, Nguyen Q. Uyen, Huynh A. Hong, Le H.and Simon M. Cutting, J. Bacteriol. 2006, 188(7)<br />
</div><br />
</div><br />
</div><br />
<br />
<h2>Biosafety group</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
Our institution (INSA Lyon) has a biosafety group. However, we have a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students follow a 4 hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training all along the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies to all the french biosafety regulations.<br />
<br/><br/><br />
As far as the legal aspect is concerned, these is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is quite restrictive, based on the precautionary principle. Even though synthetic biology doesn’t yet have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.<br />
</div><br />
</div><br />
<br />
<h2>New ideas for safety in iGEM</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
After standard parts, why not a standard chassis ?<br />
<br/><br/><br />
The INSA de Lyon team proposes that a “safety kit” should be provided to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.<br />
<br/><br/><br />
This kit should contain:<br />
<br/><br />
<ul><br />
<li>a collection of chassis that could be used to receive the DNA constructions. The strains’ genome would be the result of the gene knockout method which is used to inactivate a specific gene. With the new genetic technologies that evolve each day, now it is even possible to purchase a commercial Gene Knockout System which can be designed to knock-out the gene of interest. For example, an applications of this genetic instrument is the construction of auxotrophic strains (or nutrient-deficient) which are mutant strains of bacteria that are unable to grow on minimal media. The proliferation of these organisms outside the laboratory is limited because the lab chemicals are not found in nature.</li><br />
<br />
<li>a toxin/antitoxin system coupled with a lysing agent/cytotoxic compount/degradation agent which is provided on a separate backbone (it would leave the choice to the participants whether or not they want to use it). If the strain containing this system is released, horizontal gene transfer between a non-natural bacteria and a natural one is prohibited. Thus, with the anti-toxin inside the chassis’ genome, and the toxin and cytotoxic compound in the plasmid DNA, the horizontal gene transfer will induce the death of the recipient.<br />
</li><br />
<li>a strain suicide option represented by a gene-killer (also found on a backbone) which is activated in the absence/presence of a certain substrate. For example, Contreras <i>et al.</i> (1991) [1], proposed the construction of a confined strain capable of digesting polluted substance. In the absence of pollutant in the environment, a suicide gene is expressed and the cell is destructed.<br />
</li><br />
</ul><br />
<br/><br />
We are aware that engineering these DNA sequences is not easy. This is why we suggest that a new section or a new reward should be proposed : “ Best Safety Device”. For teams it would be a real challenge and their investment for creating new safety devices would be rewarded.<br />
<br/><br/><br />
In synthetic biology the safety and security measures are of primary importance. Every team should have a proper formation in safety and security issues. INSA de Lyon team suggests that one of the competition requirements should be a diploma validating a proper safety formation.<br />
<br/><br/><br />
<br />
<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br/><br />
1 Contreras A., Molin S, Ramos JL. 1991. Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics. Applied and Environmental Microbiology.<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/microbialControlTeam:Lyon-INSA/microbialControl2012-09-27T03:05:10Z<p>Suxiaohui: </p>
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<h2>The versatility of « Biofilm Killer »</h2><br />
<br />
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<br />
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<br />
<p>“Biofilm Killer” is designed to meet several industrial needs, because although almost every industry has problems dealing with biofilm development, cleaning biofilms does not mean the same in the different industries.</br><br />
These applications can be briefly categorized in three different needs :</br><br />
<OL><li>The need for a clean, microorganism-free, chemical-free surface. The industrial fields requiring this level of protection are basically those dealing with Human and Animal Health issues, in which the presence of contaminating microorganisms, whether good or bad, prevent certification and/or use of the instruments. This may as well concern the food industry because of health and hygiene related issues as well, although the use of a probiotic bacterium as a host for Biofilm Killer may be acceptable in certain conditions or countries.<br/><br />
<br/><li>The need for a clean deleterious microorganism-free, with a protective bacterial barrier. Two main application domains are concerned. First, in farm animal breeding, protective biofilm barriers are already in use (ex. poultry), and has application in other animal system to prevent pathogen infection and excessive mortality. The second application domains is phytoprotection, e.g. biological control of plant pathogens in agriculture. Several microbial agents have been used successfully for year to control plant diseases. In many cases the biocontrol agent is used to replace the local flora and create a protective barrier against the pathogens to prevent infection. <br/><br />
<br/><li>The need for a clean, deleterious microorganism-free but biofilm colinization-protected surface. This may apply to certain areas in which long term protection would be useful, but the presence of the protective biofilm is inadequate, e.g. food-industry, cosmetics, and other soft-chemical industry. This also apply to refrigeration systems and air-conditioning units.<br />
</OL> <p><br />
<br/><br/>The beauty of Bioflm Killer resides in its versatile design that allows the user to choose the best combination of actions for his needs. The 3 modules of Biofilm Killer are independent, and can be used efficiently in any combination with the use of non toxic inducers in very limited quantities.<br/><br />
Basically with regards to the above classification of needs the following options are suggested.<br/><br />
</br><ul><li>Health and food industry: Use of only Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and accomplish a surface 100% free of bacteria after a regular cleaning procedure steps.<br/><br />
<br/><li>Poultry and Animal raising, Oil industry : Use of Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and induction of Module 3 (STICK) to establish a protective <i>B. subtilis</i> biofilm to create a barrier flora in the intestine of the animal or on the surface of tubing to achieve long term protection against pathogen recolonization or corrosion.<br/><br />
<br/><li>Food industry, Soft chemical industry or the treatment of refrigeration units, air-condition modules : In other applications where the establishement of a bacterial biofilm cannot be envisionned, but long term protection against microbial recolonization needs to be achieved, the users can use Module 1 (Kill and Scatter) to remove the biofilm in combination with Module 2 (COAT) to generate a protective, peptide-based protective coating of the surface to protect.<br/><br/></ul><br />
<br />
To make Biofilm Killer compatible for most industrial applications, we further propose to introduce our genetic constructions in a NON-sporulant <i>Bacillus subtilis</i> strain to prevent the release and survival of this strain in the environment. (<a href="https://2012.igem.org/Team:Lyon-INSA/safety"> See safety page</a>)<br/><br/><br />
</p><br />
<br />
</div><br />
</div><br />
<br />
<h2>Focus on Oil Industry</h2><br />
<br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<div class="contenuTexte" style="width:70%;display:inline-block;"><br />
<br />
<br />
<p>Oil industry faces with important and costly difficulties due to biofilm-related issues. These include, but are not limited to, pipeline and metallic structure corrosion, porosity clogging in the rock or the tubing, microbial souring, petroleum spoilage during storage. The main consequences of microbial at the global scale are several : <br/> A reduced quality of the final product (microbial alteration), an increase in treatment costs (souring), the reduction of well production (souring, microbial alteration, clogging). Biofilm formation is involved in the corrosion of the metallic structures, including the oil-plateform, but most importantly the pipelines and tubing. <br/><br/><br />
Biofilm-induced alteration will affect the structure, resulting in two effects : <br/><br />
The first one is the clogging of the tubing, which much like cholesterol deposits in our arteries, will reduce the available circulating space, reducing the flux of gas or oil that can be transported through the pipe. Even small amounts of biofilm can negatively <br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" width="215px" style="<br />
margin-left:15px;margin-top:20px;vertical-align:top;"/><br />
</a><br />
</p><br />
<p>affect flow of hydrocarbons, as can be seen on the figure on the right showing the results of an an experiment performed on gas fluxes in presence or absence of only 8% of biofilm coverage. As a consequence of biofilm formation, we can see that about 50% of the gas flux is lost (<a href="http://www.slb.com/%7E/media/Files/resources/oilfield_review/ors12/sum12/1_microbes.pdf">from Z. Augustinovic <i>et al.</i></a>).<br/><br />
</p><br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<p>The second is the anaerobic corrosion of the metal from the structure, which will be instrumental in establishing the biofilm and induce clogging, but will also fragilize the structures.<br />
</p><br />
<center><br />
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</div><br />
<p>In the above example of biofilm-induced pipe corrosion, while a significant part of the corrosion occurs on the outside of the pipe, it is estimated that more than half of the corrosion is due to microbial growth on the inner surface of pipelines. Internal and external metallic corrosion contributes significantly to the risk of oil and gas pipeline deterioration and failure (see below), causes well and reservoir souring and plugging, and results in billions of dollars in annual costs to the oil and gas industry. <br />
</p><br />
<br />
<br />
<div class="contenuTexte" style="display:inline-block;width:60%"><br />
<p>Impact of biofilms and microbiologically influenced corrosion in oilfield. Siri plateform (center) is located in the North sea and flanked by the smaller Cecilie and Nini satellite plateforms. Seafloor lines between the 3 structures and wells carry oil and gas (gas for lift and injection water for pressure support). INSET: in 2007, water injection line ruptured. Subsequent investigation revealed a mixture of iron sulfide and other corrosion by-products plus microbes and polysaccharide slime at the rupture site. These deposits allow sulfate-reducing procaryotes and other troublesome microbes to grow protected from biocides. (Augustinovic <i>et al.</i>, Microbes- Oilfield Enemies or Allies? Oilfield Review, Summer 2012, Schlumberger)</p><br />
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"/><br />
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<br />
<p>Most importantly, failure on the production line can lead to dramatic oil or gas spills that lead to unprecedented environmental risks and damages. Such failure has recently happened on the Elgin Field, located in the North Sea ca. 240 kilometers off Aberdeen (Scotland), on a gas field exploited by the french potroleum company Total. The failure of the production line lead to a gas spill estimated to ca. 1.8 million euro loss per day for the company. Due to the location of the failure at the sea floor, it took several month for the leak to be stopped. In addition to the cost to the company, a maritime exclusion zone had to be created which perturbated maritime traffic, and ca. 20 tons of gas were released in the atmosphere daily. The previous year, in the same region an oleoduc exploited by the Royal Dutch Shell had ruptured also leading to oil spill in the North sea.<br />
</p><br />
<br />
<p>The problems with oil or gas production pipe is two folds : they are expected to be in place for decades and they often are difficult to access. Thus cleaning and locating biofilm is no simple task. Biofilms can be in dead zone which make them impossible to clean with mechanical process.</p><br />
</div><br/><br />
<br />
</div><br />
</div><br />
<br />
<h2>« Biofilm Killer »: a practical manual<br />
</h2><br />
<br />
<div class="wrapper"><br />
<br />
<div class="contenuTexte" style="width:95%;display:inline-block;"><br />
<br />
<br />
<p>Our Biofilm Killer construction in <i>Bacillus subtilis</i> 168 is designed to help address both the ease of delivery of the cleaner as well as induce a long term protection on the inner surface of the tubing. Biofilm Killer can be applied following the procedure which is already accepted in the industrial processes where Clean In Place procedures are performed. In this protocole, the process features 3 tanks usually filled with sodium carbonate, nitric acid and sodium hypochlorite. These three chemicals are used in sequence to remove the biofilm by inducing an alkaline and acidic treatment to destabilize the biofilm and a sterilizing treatment with hypochlorite. Refinements to the CIP procedure include the use of the use of specific enzymes targeting the biofilm, e.g. dispersin (Realco).<br/><br />
<P style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" width="60%" /><br />
</a></P><br />
<p><br>In order to specifically remove biofilms and to lower amount of toxic chemicals used, we propose to fill one of the CIP bulk with “Biofilm Killer”. “Biofilm Killer” will be targeted to the place to clean by the flow of water delivered in the pipe. It will swim inside the biofilm and produce and deliver in situ inside the biofilm both the biocide and the scattering agent. “Biofilm Killer” will have an action on both the target strain to kill it and on the exopolysaccharide matrix of the biofilm to dissolve it. Our physiological tests show that after 1 hour there are already significant effects of lysostaphine and dispersine. The impressive effect of the combined action of the two proteins on a staphylococcal biofilm is shown <a href="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg">HERE</a>. To maximise the dispersing effect of the construction, we recommend a 5 hour duration of “Biofilm Killer”. The scattered and killed biofilm will then be eliminated by subsequent acid, caustic and sanitizing treatment as in classical CIP, if no recolonization is required. A significant decrease in the needed amount of these chemical is expected. Alternatively, “Biofilm Killer” will be induced to colonize the surface or produce surfactin in order to form a long-term protection against deleterious recolonization of the surface.<br />
<br/><br />
Preparation and storage of Biofilm Killer is not an issue since the very same organism is already produced and stored for poultry breeding or crop plant phytoprotection. In our case, it will be most convenient to prepare Biofilm Killer as a lyophilisate since it can be stored for months until use.<br />
<br />
</p><br />
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<br />
<h2>Biofilm Killer and the Oil Patch Economy</h2><br />
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<p><br />
The oil patch is a worldwide industry and moreover a multi-billion dollars activity. It has ramifications worldwide. One can find at least one step of the production or the distribution line in every country. From the exploration to the distribution or the transformation in different products and to the consumer, this system is quite linear and can be schematized as follows : <br/><br />
<p style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" width="80%" /><br />
</a></p><br/><br />
<br />
This work flow can be divided in two types of activities : <br />
<br/><ul><li>The upstream operations concerns the production <i>sensu largo</i> of the resource itself. It includes all the operations needed for the exploration, the drilling, and the extraction necessary for the crude oil and the natural gas production. <br />
<br/><li>The downstream operations deal with the transport and transformation of the product itself. It includes transporting the oil or gas by pipes or Tankers, the refining, the retailing and the consuming, until it finally reaches the consumer, which can be the car owner at the gas station or a company using petroleum-derived products.<br />
</ul><br />
<br/>The gas and oil patch is the biggest industry in the world. The daily production of oil is ca. 13 billion liters. It is dominated by 5 or 6, extremely big companies called “Supermajors” which rule the system and have control over it: among them are BP, Total or Shell. Despite their gigantic size, these companies control only ca. 5% of the oil reserves worldwide. The large majority of the reserve is controlled by local and national companies such as the Saudi Aramco, the iranian National Iranian Oil Company or the koweti Kuwait Petroleum Corporation.<br />
<br/><br/><br />
Oil or gas production involves millions of kilometers of tubing at the production site or for transportation, tanks and tankers for storage and oversea transportation. Moreover, oil extraction often involves the use of large amounts of water, which create a very favorable environments for microbes to grow in. This oil and water mixture travels thourgh the pipe until it is finally separated at the surface. Thus, oil propduction represent a vast numbers of tanks and kilometers of pipes in which micrbial biofilms are likely to grow and induce corrosion, and which need to be cleaned!<br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png" width="5%" style="float:left;" /><div style="width:800px;float:right;margin-right:10px">This logo means that our solution “Biofilm Killer” can be used in this part of the oil process to remove bacteria and clean pipes or tanks and save the company from much bigger issues, such as a hole in a pipe letting gas or oil flow out in the deep of an ocean, meaning loss of millions dollars.</div><br/><br />
<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/microbialControlTeam:Lyon-INSA/microbialControl2012-09-27T03:04:10Z<p>Suxiaohui: </p>
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<h2>The versatility of « Biofilm Killer »</h2><br />
<br />
<div class="wrapper"><br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<br />
<p>“Biofilm Killer” is designed to meet several industrial needs, because although almost every industry has problems dealing with biofilm development, cleaning biofilms does not mean the same in the different industries.</br><br />
These applications can be briefly categorized in three different needs :</br><br />
<OL><li>The need for a clean, microorganism-free, chemical-free surface. The industrial fields requiring this level of protection are basically those dealing with Human and Animal Health issues, in which the presence of contaminating microorganisms, whether good or bad, prevent certification and/or use of the instruments. This may as well concern the food industry because of health and hygiene related issues as well, although the use of a probiotic bacterium as a host for Biofilm Killer may be acceptable in certain conditions or countries.<br/><br />
<br/><li>The need for a clean deleterious microorganism-free, with a protective bacterial barrier. Two main application domains are concerned. First, in farm animal breeding, protective biofilm barriers are already in use (ex. poultry), and has application in other animal system to prevent pathogen infection and excessive mortality. The second application domains is phytoprotection, e.g. biological control of plant pathogens in agriculture. Several microbial agents have been used successfully for year to control plant diseases. In many cases the biocontrol agent is used to replace the local flora and create a protective barrier against the pathogens to prevent infection. <br/><br />
<br/><li>The need for a clean, deleterious microorganism-free but biofilm colinization-protected surface. This may apply to certain areas in which long term protection would be useful, but the presence of the protective biofilm is inadequate, e.g. food-industry, cosmetics, and other soft-chemical industry. This also apply to refrigeration systems and air-conditioning units.<br />
</OL> <p><br />
<br/><br/>The beauty of Bioflm Killer resides in its versatile design that allows the user to choose the best combination of actions for his needs. The 3 modules of Biofilm Killer are independent, and can be used efficiently in any combination with the use of non toxic inducers in very limited quantities.<br/><br />
Basically with regards to the above classification of needs the following options are suggested.<br/><br />
</br><ul><li>Health and food industry: Use of only Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and accomplish a surface 100% free of bacteria after a regular cleaning procedure steps.<br/><br />
<br/><li>Poultry and Animal raising, Oil industry : Use of Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and induction of Module 3 (STICK) to establish a protective <i>B. subtilis</i> biofilm to create a barrier flora in the intestine of the animal or on the surface of tubing to achieve long term protection against pathogen recolonization or corrosion.<br/><br />
<br/><li>Food industry, Soft chemical industry or the treatment of refrigeration units, air-condition modules : In other applications where the establishement of a bacterial biofilm cannot be envisionned, but long term protection against microbial recolonization needs to be achieved, the users can use Module 1 (Kill and Scatter) to remove the biofilm in combination with Module 2 (COAT) to generate a protective, peptide-based protective coating of the surface to protect.<br/><br/></ul><br />
<br />
To make Biofilm Killer compatible for most industrial applications, we further propose to introduce our genetic constructions in a NON-sporulant <i>Bacillus subtilis</i> strain to prevent the release and survival of this strain in the environment. (<a href="https://2012.igem.org/Team:Lyon-INSA/safety"> See safety page</a>)<br/><br/><br />
</p><br />
<br />
</div><br />
</div><br />
<br />
<h2>Focus on Oil Industry</h2><br />
<br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<div class="contenuTexte" style="width:70%;display:inline-block;"><br />
<br />
<br />
<p>Oil industry faces with important and costly difficulties due to biofilm-related issues. These include, but are not limited to, pipeline and metallic structure corrosion, porosity clogging in the rock or the tubing, microbial souring, petroleum spoilage during storage. The main consequences of microbial at the global scale are several : <br/> A reduced quality of the final product (microbial alteration), an increase in treatment costs (souring), the reduction of well production (souring, microbial alteration, clogging). Biofilm formation is involved in the corrosion of the metallic structures, including the oil-plateform, but most importantly the pipelines and tubing. <br/><br/><br />
Biofilm-induced alteration will affect the structure, resulting in two effects : <br/><br />
The first one is the clogging of the tubing, which much like cholesterol deposits in our arteries, will reduce the available circulating space, reducing the flux of gas or oil that can be transported through the pipe. Even small amounts of biofilm can negatively <br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" width="215px" style="<br />
margin-left:15px;margin-top:20px;vertical-align:top;"/><br />
</a><br />
</p><br />
<p>affect flow of hydrocarbons, as can be seen on the figure on the right showing the results of an an experiment performed on gas fluxes in presence or absence of only 8% of biofilm coverage. As a consequence of biofilm formation, we can see that about 50% of the gas flux is lost (<a href="http://www.slb.com/%7E/media/Files/resources/oilfield_review/ors12/sum12/1_microbes.pdf">from Z. Augustinovic <i>et al.</i></a>).<br/><br />
</p><br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<p>The second is the anaerobic corrosion of the metal from the structure, which will be instrumental in establishing the biofilm and induce clogging, but will also fragilize the structures.<br />
</p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2012/b/be/Tube-b.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/Tube-b.png" width="300px" style="<br />
margin-left:20px;margin-top:20px;vertical-align:top;"/></a></center><br/><br />
</div><br />
<p>In the above example of biofilm-induced pipe corrosion, while a significant part of the corrosion occurs on the outside of the pipe, it is estimated that more than half of the corrosion is due to microbial growth on the inner surface of pipelines. Internal and external metallic corrosion contributes significantly to the risk of oil and gas pipeline deterioration and failure (see below), causes well and reservoir souring and plugging, and results in billions of dollars in annual costs to the oil and gas industry. <br />
</p><br />
<br />
<br />
<div class="contenuTexte" style="display:inline-block;width:60%"><br />
<p>Impact of biofilms and microbiologically influenced corrosion in oilfield. Siri plateform (center) is located in the North sea and flanked by the smaller Cecilie and Nini satellite plateforms. Seafloor lines between the 3 structures and wells carry oil and gas (gas for lift and injection water for pressure support). INSET: in 2007, water injection line ruptured. Subsequent investigation revealed a mixture of iron sulfide and other corrosion by-products plus microbes and polysaccharide slime at the rupture site. These deposits allow sulfate-reducing procaryotes and other troublesome microbes to grow protected from biocides. (Augustinovic <i>et al.</i>, Microbes- Oilfield Enemies or Allies? Oilfield Review, Summer 2012, Schlumberger)</p><br />
<br/><br/><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/e/e2/Oilindus.gif" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e2/Oilindus.gif" width="30%" style="<br />
margin-left:20px;margin-top:0px;vertical-align:top;"/><br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/c/cb/Industrya.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Industrya.png" width="300px" style="margin:20px;vertical-align:top;margin-top:40px;<br />
"/><br />
</a><br />
<br />
<div class="contenuTexte" style="display:inline-block;width:60%;"><br />
<br />
<p>Most importantly, failure on the production line can lead to dramatic oil or gas spills that lead to unprecedented environmental risks and damages. Such failure has recently happened on the Elgin Field, located in the North Sea ca. 240 kilometers off Aberdeen (Scotland), on a gas field exploited by the french potroleum company Total. The failure of the production line lead to a gas spill estimated to ca. 1.8 million euro loss per day for the company. Due to the location of the failure at the sea floor, it took several month for the leak to be stopped. In addition to the cost to the company, a maritime exclusion zone had to be created which perturbated maritime traffic, and ca. 20 tons of gas were released in the atmosphere daily. The previous year, in the same region an oleoduc exploited by the Royal Dutch Shell had ruptured also leading to oil spill in the North sea.<br />
</p><br />
<br />
<p>The problems with oil or gas production pipe is two folds : they are expected to be in place for decades and they often are difficult to access. Thus cleaning and locating biofilm is no simple task. Biofilms can be in dead zone which make them impossible to clean with mechanical process.</p><br />
</div><br/><br />
<br />
</div><br />
</div><br />
<br />
<h2>« Biofilm Killer »: a practical manual<br />
</h2><br />
<br />
<div class="wrapper"><br />
<br />
<div class="contenuTexte" style="width:95%;display:inline-block;"><br />
<br />
<br />
<p>Our Biofilm Killer construction in <i>Bacillus subtilis</i> 168 is designed to help address both the ease of delivery of the cleaner as well as induce a long term protection on the inner surface of the tubing. Biofilm Killer can be applied following the procedure which is already accepted in the industrial processes where Clean In Place procedures are performed. In this protocole, the process features 3 tanks usually filled with sodium carbonate, nitric acid and sodium hypochlorite. These three chemicals are used in sequence to remove the biofilm by inducing an alkaline and acidic treatment to destabilize the biofilm and a sterilizing treatment with hypochlorite. Refinements to the CIP procedure include the use of the use of specific enzymes targeting the biofilm, e.g. dispersin (Realco).<br/><br />
<P style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" width="60%" /><br />
</a></P><br />
<p><br>In order to specifically remove biofilms and to lower amount of toxic chemicals used, we propose to fill one of the CIP bulk with “Biofilm Killer”. “Biofilm Killer” will be targeted to the place to clean by the flow of water delivered in the pipe. It will swim inside the biofilm and produce and deliver in situ inside the biofilm both the biocide and the scattering agent. “Biofilm Killer” will have an action on both the target strain to kill it and on the exopolysaccharide matrix of the biofilm to dissolve it. Our physiological tests show that after 1 hour there are already significant effects of lysostaphine and dispersine. The impressive effect of the combined action of the two proteins on a staphylococcal biofilm is exemplified <a href="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg">HERE</a>. To maximise the dispersing effect of the construction, we recommend a 5 hour duration of “Biofilm Killer”. The scattered and killed biofilm will then be eliminated by subsequent acid, caustic and sanitizing treatment as in classical CIP, if no recolonization is required. A significant decrease in the needed amount of these chemical is expected. Alternatively, “Biofilm Killer” will be induced to colonize the surface or produce surfactin in order to form a long-term protection against deleterious recolonization of the surface.<br />
<br/><br />
Preparation and storage of Biofilm Killer is not an issue since the very same organism is already produced and stored for poultry breeding or crop plant phytoprotection. In our case, it will be most convenient to prepare Biofilm Killer as a lyophilisate since it can be stored for months until use.<br />
<br />
</p><br />
<br />
<br />
</div><br />
</div><br />
<br />
<br />
<h2>Biofilm Killer and the Oil Patch Economy</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte" style="width:95%;display:inline-block;"><br />
<p><br />
The oil patch is a worldwide industry and moreover a multi-billion dollars activity. It has ramifications worldwide. One can find at least one step of the production or the distribution line in every country. From the exploration to the distribution or the transformation in different products and to the consumer, this system is quite linear and can be schematized as follows : <br/><br />
<p style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" width="80%" /><br />
</a></p><br/><br />
<br />
This work flow can be divided in two types of activities : <br />
<br/><ul><li>The upstream operations concerns the production <i>sensu largo</i> of the resource itself. It includes all the operations needed for the exploration, the drilling, and the extraction necessary for the crude oil and the natural gas production. <br />
<br/><li>The downstream operations deal with the transport and transformation of the product itself. It includes transporting the oil or gas by pipes or Tankers, the refining, the retailing and the consuming, until it finally reaches the consumer, which can be the car owner at the gas station or a company using petroleum-derived products.<br />
</ul><br />
<br/>The gas and oil patch is the biggest industry in the world. The daily production of oil is ca. 13 billion liters. It is dominated by 5 or 6, extremely big companies called “Supermajors” which rule the system and have control over it: among them are BP, Total or Shell. Despite their gigantic size, these companies control only ca. 5% of the oil reserves worldwide. The large majority of the reserve is controlled by local and national companies such as the Saudi Aramco, the iranian National Iranian Oil Company or the koweti Kuwait Petroleum Corporation.<br />
<br/><br/><br />
Oil or gas production involves millions of kilometers of tubing at the production site or for transportation, tanks and tankers for storage and oversea transportation. Moreover, oil extraction often involves the use of large amounts of water, which create a very favorable environments for microbes to grow in. This oil and water mixture travels thourgh the pipe until it is finally separated at the surface. Thus, oil propduction represent a vast numbers of tanks and kilometers of pipes in which micrbial biofilms are likely to grow and induce corrosion, and which need to be cleaned!<br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png" width="5%" style="float:left;" /><div style="width:800px;float:right;margin-right:10px">This logo means that our solution “Biofilm Killer” can be used in this part of the oil process to remove bacteria and clean pipes or tanks and save the company from much bigger issues, such as a hole in a pipe letting gas or oil flow out in the deep of an ocean, meaning loss of millions dollars.</div><br/><br />
<br />
<p><br />
</div><br />
</div><br />
</div><br />
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</body><br />
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</html><br />
{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/microbialControlTeam:Lyon-INSA/microbialControl2012-09-27T03:03:17Z<p>Suxiaohui: </p>
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<div class="boutonOnglet " onclick="window.location='BiofilmK';">"Biofilm Killer"</div><br />
<div class="boutonOnglet BOClicked" >Applications</div><br />
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<h1>Applications</h1><br />
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<h2>The versatility of « Biofilm Killer »</h2><br />
<br />
<div class="wrapper"><br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<br />
<p>“Biofilm Killer” is designed to meet several industrial needs, because although almost every industry has problems dealing with biofilm development, cleaning biofilms does not mean the same in the different industries.</br><br />
These applications can be briefly categorized in three different needs :</br><br />
<OL><li>The need for a clean, microorganism-free, chemical-free surface. The industrial fields requiring this level of protection are basically those dealing with Human and Animal Health issues, in which the presence of contaminating microorganisms, whether good or bad, prevent certification and/or use of the instruments. This may as well concern the food industry because of health and hygiene related issues as well, although the use of a probiotic bacterium as a host for Biofilm Killer may be acceptable in certain conditions or countries.<br/><br />
<br/><li>The need for a clean deleterious microorganism-free, with a protective bacterial barrier. Two main application domains are concerned. First, in farm animal breeding, protective biofilm barriers are already in use (ex. poultry), and has application in other animal system to prevent pathogen infection and excessive mortality. The second application domains is phytoprotection, e.g. biological control of plant pathogens in agriculture. Several microbial agents have been used successfully for year to control plant diseases. In many cases the biocontrol agent is used to replace the local flora and create a protective barrier against the pathogens to prevent infection. <br/><br />
<br/><li>The need for a clean, deleterious microorganism-free but biofilm colinization-protected surface. This may apply to certain areas in which long term protection would be useful, but the presence of the protective biofilm is inadequate, e.g. food-industry, cosmetics, and other soft-chemical industry. This also apply to refrigeration systems and air-conditioning units.<br />
</OL> <p><br />
<br/><br/>The beauty of Bioflm Killer resides in its versatile design that allows the user to choose the best combination of actions for his needs. The 3 modules of Biofilm Killer are independent, and can be used efficiently in any combination with the use of non toxic inducers in very limited quantities.<br/><br />
Basically with regards to the above classification of needs the following options are suggested.<br/><br />
</br><ul><li>Health and food industry: Use of only Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and accomplish a surface 100% free of bacteria after a regular cleaning procedure steps.<br/><br />
<br/><li>Poultry and Animal raising, Oil industry : Use of Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and induction of Module 3 (STICK) to establish a protective <i>B. subtilis</i> biofilm to create a barrier flora in the intestine of the animal or on the surface of tubing to achieve long term protection against pathogen recolonization or corrosion.<br/><br />
<br/><li>Food industry, Soft chemical industry or the treatment of refrigeration units, air-condition modules : In other applications where the establishement of a bacterial biofilm cannot be envisionned, but long term protection against microbial recolonization needs to be achieved, the users can use Module 1 (Kill and Scatter) to remove the biofilm in combination with Module 2 (COAT) to generate a protective, peptide-based protective coating of the surface to protect.<br/><br/></ul><br />
<br />
To make Biofilm Killer compatible for most industrial applications, we further propose to introduce our genetic constructions in a NON-sporulant <i>Bacillus subtilis</i> strain to prevent the release and survival of this strain in the environment. (<a href="https://2012.igem.org/Team:Lyon-INSA/safety" See safety page</a>)<br/><br/><br />
</p><br />
<br />
</div><br />
</div><br />
<br />
<h2>Focus on Oil Industry</h2><br />
<br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
<br />
<div class="contenuTexte" style="width:70%;display:inline-block;"><br />
<br />
<br />
<p>Oil industry faces with important and costly difficulties due to biofilm-related issues. These include, but are not limited to, pipeline and metallic structure corrosion, porosity clogging in the rock or the tubing, microbial souring, petroleum spoilage during storage. The main consequences of microbial at the global scale are several : <br/> A reduced quality of the final product (microbial alteration), an increase in treatment costs (souring), the reduction of well production (souring, microbial alteration, clogging). Biofilm formation is involved in the corrosion of the metallic structures, including the oil-plateform, but most importantly the pipelines and tubing. <br/><br/><br />
Biofilm-induced alteration will affect the structure, resulting in two effects : <br/><br />
The first one is the clogging of the tubing, which much like cholesterol deposits in our arteries, will reduce the available circulating space, reducing the flux of gas or oil that can be transported through the pipe. Even small amounts of biofilm can negatively <br />
</div><br />
<a href="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e6/Flow.png" width="215px" style="<br />
margin-left:15px;margin-top:20px;vertical-align:top;"/><br />
</a><br />
</p><br />
<p>affect flow of hydrocarbons, as can be seen on the figure on the right showing the results of an an experiment performed on gas fluxes in presence or absence of only 8% of biofilm coverage. As a consequence of biofilm formation, we can see that about 50% of the gas flux is lost (<a href="http://www.slb.com/%7E/media/Files/resources/oilfield_review/ors12/sum12/1_microbes.pdf">from Z. Augustinovic <i>et al.</i></a>).<br/><br />
</p><br />
<br />
<div class="contenuTexte" style="display:inline-block;"><br />
<p>The second is the anaerobic corrosion of the metal from the structure, which will be instrumental in establishing the biofilm and induce clogging, but will also fragilize the structures.<br />
</p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2012/b/be/Tube-b.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/b/be/Tube-b.png" width="300px" style="<br />
margin-left:20px;margin-top:20px;vertical-align:top;"/></a></center><br/><br />
</div><br />
<p>In the above example of biofilm-induced pipe corrosion, while a significant part of the corrosion occurs on the outside of the pipe, it is estimated that more than half of the corrosion is due to microbial growth on the inner surface of pipelines. Internal and external metallic corrosion contributes significantly to the risk of oil and gas pipeline deterioration and failure (see below), causes well and reservoir souring and plugging, and results in billions of dollars in annual costs to the oil and gas industry. <br />
</p><br />
<br />
<br />
<div class="contenuTexte" style="display:inline-block;width:60%"><br />
<p>Impact of biofilms and microbiologically influenced corrosion in oilfield. Siri plateform (center) is located in the North sea and flanked by the smaller Cecilie and Nini satellite plateforms. Seafloor lines between the 3 structures and wells carry oil and gas (gas for lift and injection water for pressure support). INSET: in 2007, water injection line ruptured. Subsequent investigation revealed a mixture of iron sulfide and other corrosion by-products plus microbes and polysaccharide slime at the rupture site. These deposits allow sulfate-reducing procaryotes and other troublesome microbes to grow protected from biocides. (Augustinovic <i>et al.</i>, Microbes- Oilfield Enemies or Allies? Oilfield Review, Summer 2012, Schlumberger)</p><br />
<br/><br/><br />
</div><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/e/e2/Oilindus.gif" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e2/Oilindus.gif" width="30%" style="<br />
margin-left:20px;margin-top:0px;vertical-align:top;"/><br />
</a><br />
<br />
<a href="https://static.igem.org/mediawiki/2012/c/cb/Industrya.png" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cb/Industrya.png" width="300px" style="margin:20px;vertical-align:top;margin-top:40px;<br />
"/><br />
</a><br />
<br />
<div class="contenuTexte" style="display:inline-block;width:60%;"><br />
<br />
<p>Most importantly, failure on the production line can lead to dramatic oil or gas spills that lead to unprecedented environmental risks and damages. Such failure has recently happened on the Elgin Field, located in the North Sea ca. 240 kilometers off Aberdeen (Scotland), on a gas field exploited by the french potroleum company Total. The failure of the production line lead to a gas spill estimated to ca. 1.8 million euro loss per day for the company. Due to the location of the failure at the sea floor, it took several month for the leak to be stopped. In addition to the cost to the company, a maritime exclusion zone had to be created which perturbated maritime traffic, and ca. 20 tons of gas were released in the atmosphere daily. The previous year, in the same region an oleoduc exploited by the Royal Dutch Shell had ruptured also leading to oil spill in the North sea.<br />
</p><br />
<br />
<p>The problems with oil or gas production pipe is two folds : they are expected to be in place for decades and they often are difficult to access. Thus cleaning and locating biofilm is no simple task. Biofilms can be in dead zone which make them impossible to clean with mechanical process.</p><br />
</div><br/><br />
<br />
</div><br />
</div><br />
<br />
<h2>« Biofilm Killer »: a practical manual<br />
</h2><br />
<br />
<div class="wrapper"><br />
<br />
<div class="contenuTexte" style="width:95%;display:inline-block;"><br />
<br />
<br />
<p>Our Biofilm Killer construction in <i>Bacillus subtilis</i> 168 is designed to help address both the ease of delivery of the cleaner as well as induce a long term protection on the inner surface of the tubing. Biofilm Killer can be applied following the procedure which is already accepted in the industrial processes where Clean In Place procedures are performed. In this protocole, the process features 3 tanks usually filled with sodium carbonate, nitric acid and sodium hypochlorite. These three chemicals are used in sequence to remove the biofilm by inducing an alkaline and acidic treatment to destabilize the biofilm and a sterilizing treatment with hypochlorite. Refinements to the CIP procedure include the use of the use of specific enzymes targeting the biofilm, e.g. dispersin (Realco).<br/><br />
<P style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" class="fancyable"><br />
<img src="https://static.igem.org/mediawiki/2012/1/11/CIP.JPG" width="60%" /><br />
</a></P><br />
<p><br>In order to specifically remove biofilms and to lower amount of toxic chemicals used, we propose to fill one of the CIP bulk with “Biofilm Killer”. “Biofilm Killer” will be targeted to the place to clean by the flow of water delivered in the pipe. It will swim inside the biofilm and produce and deliver in situ inside the biofilm both the biocide and the scattering agent. “Biofilm Killer” will have an action on both the target strain to kill it and on the exopolysaccharide matrix of the biofilm to dissolve it. Our physiological tests show that after 1 hour there are already significant effects of lysostaphine and dispersine. The impressive effect of the combined action of the two proteins on a staphylococcal biofilm is exemplified <a href="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg">HERE</a>. To maximise the dispersing effect of the construction, we recommend a 5 hour duration of “Biofilm Killer”. The scattered and killed biofilm will then be eliminated by subsequent acid, caustic and sanitizing treatment as in classical CIP, if no recolonization is required. A significant decrease in the needed amount of these chemical is expected. Alternatively, “Biofilm Killer” will be induced to colonize the surface or produce surfactin in order to form a long-term protection against deleterious recolonization of the surface.<br />
<br/><br />
Preparation and storage of Biofilm Killer is not an issue since the very same organism is already produced and stored for poultry breeding or crop plant phytoprotection. In our case, it will be most convenient to prepare Biofilm Killer as a lyophilisate since it can be stored for months until use.<br />
<br />
</p><br />
<br />
<br />
</div><br />
</div><br />
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<h2>Biofilm Killer and the Oil Patch Economy</h2><br />
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The oil patch is a worldwide industry and moreover a multi-billion dollars activity. It has ramifications worldwide. One can find at least one step of the production or the distribution line in every country. From the exploration to the distribution or the transformation in different products and to the consumer, this system is quite linear and can be schematized as follows : <br/><br />
<p style="text-align:center"><a href="https://static.igem.org/mediawiki/2012/6/65/Image_analyse_oil_patch.jpg" class="fancyable"><br />
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This work flow can be divided in two types of activities : <br />
<br/><ul><li>The upstream operations concerns the production <i>sensu largo</i> of the resource itself. It includes all the operations needed for the exploration, the drilling, and the extraction necessary for the crude oil and the natural gas production. <br />
<br/><li>The downstream operations deal with the transport and transformation of the product itself. It includes transporting the oil or gas by pipes or Tankers, the refining, the retailing and the consuming, until it finally reaches the consumer, which can be the car owner at the gas station or a company using petroleum-derived products.<br />
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<br/>The gas and oil patch is the biggest industry in the world. The daily production of oil is ca. 13 billion liters. It is dominated by 5 or 6, extremely big companies called “Supermajors” which rule the system and have control over it: among them are BP, Total or Shell. Despite their gigantic size, these companies control only ca. 5% of the oil reserves worldwide. The large majority of the reserve is controlled by local and national companies such as the Saudi Aramco, the iranian National Iranian Oil Company or the koweti Kuwait Petroleum Corporation.<br />
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Oil or gas production involves millions of kilometers of tubing at the production site or for transportation, tanks and tankers for storage and oversea transportation. Moreover, oil extraction often involves the use of large amounts of water, which create a very favorable environments for microbes to grow in. This oil and water mixture travels thourgh the pipe until it is finally separated at the surface. Thus, oil propduction represent a vast numbers of tanks and kilometers of pipes in which micrbial biofilms are likely to grow and induce corrosion, and which need to be cleaned!<br/><br />
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Logo_amst%C3%A9rix_page_accueil.png" width="5%" style="float:left;" /><div style="width:800px;float:right;margin-right:10px">This logo means that our solution “Biofilm Killer” can be used in this part of the oil process to remove bacteria and clean pipes or tanks and save the company from much bigger issues, such as a hole in a pipe letting gas or oil flow out in the deep of an ocean, meaning loss of millions dollars.</div><br/><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/collaborationTeam:Lyon-INSA/collaboration2012-09-27T02:58:15Z<p>Suxiaohui: </p>
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<h1>How did we collaborate?</h1><br />
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Collaboration is a very important issue. We think that sharing our ideas could be very beneficial for both teams engaged in the collaboration. There are more than 240 teams involved in the iGEM 2012 so it was impossible not to find another team sharing some aspects of our project.<br><br />
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<h3>iGEM FRANCE</h3><br />
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<a href="http://sites.google.com/site/igemfrance/"><font>iGEM FRANCE</font></a> is a collaborative website that gathers the french iGEM community together. It has been created in the beginning of 2012 by Paris-Bettencourt team as a help for students and professors to create and run their iGEM team. The website is above all a way to share knowledge, either through its forum, or by arranging meetings. <br><br />
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<h3>Collaboration with the University of British Columbia (UBC)</h3><br />
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We have established a collaboration with UBC for our human practices. Indeed UBC is working on Intellectual property (IP) this year, an aspect we also chose to explore this year.<br><br />
One of our advisors is a Professor in Economics. Thus, we have suggested to UBC when they sent their survey on IP, that we could share with them our knowledge and thoughts to improve their survey or to interpret the results of their survey. In exchange they shared the results of their survey with us.<br><br />
We have used their results to show what kind of IP issues iGEM teams could meet.<br />
To see the results of our collaboration you might want to read our <a href="https://2012.igem.org/Team:Lyon-INSA/humanPractice"><font> human practice </font></a>.<br><br />
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<h3>An interesting way to undertake a collaboration with Goettingen team</h3><br />
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Furthermore, we have tried to begin a collaboration with the Goettingen team. They are working on a super swimmer <i>E. coli</i> strain. We have proposed them different ways for collaboration. For example, we were interested in comparing the impact of swimming on biofilm destabilization between their <i>E. coli</i> strain and our <i>Bacillus subtilis</i> natural swimmer strain. We offered to send them our <i>Bacillus subtilis</i> strain to also make them able to perform comparative swimming behavior studies between both strains. Several different reasons made this collaboration unsuccessful.<br><br />
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<h3>Collaboration with Munich</h3><br />
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We took part in Munich's survey about their proposition of standardization of BioBricks part descriptions.<br><br />
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<center><img id="TUM12_badge" width="150px" src="https://static.igem.org/mediawiki/2012/c/c6/TUM12_Collaboration_medal1.png" alt="We completed TU Munich's survey on Standardization of BioBrick part descriptions"/></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/videosTeam:Lyon-INSA/videos2012-09-27T01:46:38Z<p>Suxiaohui: </p>
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<br>We are glad to present you the Lyon-INSA 2012 iGEM team. <br><br><br />
<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtwcrk?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtwcrk_lyon-insa-2012-igem-team_tech" target="_blank">Lyon INSA 2012 iGEM team</a> </center><br><br><br />
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This 2012 iGEM competition was a very amazing experience, and we would like to share our journey with you.<br><br><br />
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<center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtwcp8?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtwcp8_mon-film-part2-fin_tech" target="_blank">Journey of Lyon INSA 2012</a></center><br><br><br />
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If you haven't already seen the project description, this is how Bacillix is working !<br><br><br />
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<center><center><iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xtsdia?logo=0"></iframe><br /><a href="http://www.dailymotion.com/video/xtsdia_our-project-presentation-biofilm-killer-lyon-insa-igem_tech" target="_blank"><font color ="purple">Our project presentation Biofilm Killer Lyon...</font></a></center></center><br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/datapageTeam:Lyon-INSA/datapage2012-09-27T01:41:47Z<p>Suxiaohui: </p>
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<h1>Interactive pattern of our construction : Toggle switch option</h1><br />
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<h1>Our parts : </h1><br />
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<div class="petitSsTitre">Data about our favorite new parts </div><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802001" target="_blank"><font color="#664499"><b>1. Main Page</b></font></a>: <b>Dispersin generator for <i>B. subtilis</i></b> BBa_K802001 : This part associates the <i>Bacillus subtilis</i> constitutive promoter (P<sub>veg</sub>) with the dispersin B gene (<i>dspB</i>). This part allows to efficiently scatter Staphylococci biofilms. <br><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802003" target="_blank"><font color="#664499"><b>2. Main Page</b></font></a>: <b>Shuttle vector for <i>E. coli</i> and <i>B. subtilis</i> </b>BBa_K802003 : This part is a shuttle vector of 6.5kb which is Ampicillin resistant in <i>E. coli</i> and Erythromycin resistant in <i>B. subtilis</i>. It is a <b>low</b> copy number plasmid in <i>B. subtilis</i> and a high copy number plasmid in <i>E. coli</i>. It has a polylinker containing all 4 of the iGEM restriction sites. This vector is characterized in <i>B. subtilis</i> by a transformation yield of 70 transformants/µg and a erythromycin resistance up to 900 µg/mL.<br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802000" target="_blank"><font color="#664499"><b>3. Main Page</b></font></a>: <b>Lysostaphin generator for <i>B. subtilis</i></b> BBa_K802000 : This part associates the <i>Bacillus subtilis</i> constitutive promoter (P<sub>veg</sub>) with the lysostaphin gene. This part allows an efficient killing of <i>S. aureus</i> cells.<br><br />
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<div class="petitSsTitre">We've also characterized the following new parts </div><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802002" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>P<sub>lac</sub>-RBS-GFP</b> BBa_K802002 : This part has been designed to determine the behavior of the P<sub>lac</sub> promoter used to drive the STICK module (<span class="unProto" onclick="window.open('http://partsregistry.org/Part:BBa_K802009', 'Part BBa_K802009'); return false;">Part BBa_K802009</span>)<br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802004" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>Shuttle vector for <i>E. coli</i> and <i>B. subtilis</i> </b> BBa_K802004 : This part is a shuttle vector of 6.5kb which is Ampicillin resistant in <i>E. coli</i> and Erythromycin resistant in <i>B. subtilis</i>. It is a <b>high</b> copy number plasmid in both <i>B. subtilis</i> and <i>E. coli</i>. It has a polylinker containing all 4 of the iGEM restriction sites. This vector is characterized in <i>B. subtilis</i> by a transformation yield of 80 transformants/µg and a erythromycin resistance to at least 1.5 mg/mL.<br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K802009" target="_blank"><font color="#664499"><b>Main Page</b></font></a>: <b>Surfactin generator and biofilm repressor for <i>B. subtilis</i></b> BBa_K802009: This part can be used to induce surfactin production and to repress the biofilm formation in <i> B. subtilis</i> strains (COAT module). <br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/safetyTeam:Lyon-INSA/safety2012-09-27T01:26:35Z<p>Suxiaohui: </p>
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<h1>Safety</h1><br />
<div class="contenuTexte introduction" style="margin:20px"><br />
We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified <i>Bacillus subtilis</i> strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked also with an <i>Escherichia coli</i> strain and as a biofilm model with <i>Staphylococcus epidermidis</i>. <br />
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<h2>Researcher/Public/Environmental Safety</h2><br />
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Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their toxicity for example. For these reasons: the access of the laboratory was limited to those involved in the project and all work benches were cleaned every day and the whole lab cleaned every week.<br />
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CLEAN AREAS TO ENCOURAGE GOOD PRACTICES<br />
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Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider the fact of having a proper training in safety and security at the beginning of our experimental work prepared us to be more organised, responsible for our actions and respectful for those of others. To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.<br />
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<strong>Global hazards and risks :</strong> Electricity and gas: we have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. Personal protective equipement, including appropriate labcoats, gloves and safety glasses were worn for lab experiments. No one was allowed to work alone in the laboratory. In addition, the french system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life saving diploma and are also trained for firefighting.<br />
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<strong>Main chemical hazards and risks :</strong> Most of the reagents used are irritant, toxic and can be potential carcinogens (agarose, polyacrylamid, methanol, Ethidium bromide…)<br />
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To minimize the impact of their use, they are manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials are clearly identified/labeled to avoid inappropriate mix between two non compatible solvents. All reagents are eliminated in the appropriate waste recovered barrels.<br />
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We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates whithin the double strand DNA helix. Many biological processes can thus be affected such as transcription and replication. To avoid the dissemination of EtBr in the lab, it is stored and used in the same room where the electrophoresis gels are revealed. EtBr is NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. This room is locked by a key. The access to this room is forbidden to any person who does not carry a lab coat. Dedicated gloves are used for EtBr manipulation and must not leave the room. They are discarded in a specific trash barrel for genotoxines contaminated material to gather with stained gels. EtBr-contaminated material is further processed and decontaminated by an external service.<br />
<br/><br/><br />
Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.<br />
<br/><br/><br />
<strong>Main biological hazards and risks:</strong> All <i>Bacillus subtilis</i>, <i>Echerichia coli</i> and <i>Staphylococcus epidermidis</i> strains we used have a biosafety level of 1, which means they are not known to cause disease and have minimal environmental hazards.<br />
<br/><br/><br />
At this level the precautions concerning the lab-material are minimal: wearing gloves and face protection when needed. Any contaminated material is discarded in a trash can to be autoclaved within 2 days. The decontamination procedure is similar to any other applied to frequently encountered virus or bacteria (ex: washing hands with antibacterial soap, disinfecting any surface in the laboratory that has been exposed). Our work benches are decontaminated with ethanol 70% after each manipulation. And contaminated media and materials are autoclaved at least every 2 days to avoid any release in environment.<br />
<br/><br/><br />
All the students must have their vaccinations up to date. In case a student harbors an injury, it must be covered so it is not exposed to the bacteria. All rooms are equipped with with a first aid kit.<br />
In the building where the labwork was performed, no laboratory is manipulating pathogenic microorganisms, which limits the risks to students, and the recombination possibility between a pathogenic bacteria and the lab strains.<br />
<br/><br/><br />
For better protection the following basic safety rules applied for all students during lab work:<br/><br />
<ul><br />
<li> Be aware of the risks and hazards involved in any experiment</li><br />
<br />
<li>Know where the fire extinguisher, safety shower and general electrical circuit breaker are located in each room</li><br />
<br />
<li>No food or drink in the laboratory</li><br />
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<li>Wear a buttoned up lab coat</li><br />
<br />
<li> Long hair need to be tied back</li><br />
<br />
<li>Contact lenses are forbidden except if people wear a face protection or safety glasses</li><br />
<br />
<li>Spillage needs to be cleaned up immediately</li><br />
<br />
<li>Lab benches are cleaned after each experiment</li><br />
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<li>Technical and safety files are available near all the lab equipments</li><br />
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<li>No chemical touching, sniffing or tasting</li><br />
<br />
<li>No mouth-pipeting</li><br />
<br />
<li>No returning unused chemicals to their containers</li><br />
<br />
<li>No hazardous material disposal down the drain</li><br />
</ul><br />
<br/><br />
This specific localization in a public research laboratory building allows us to benefit from researcher and student law safety (or under application in France) which are mandatory in each lab in France. For example, we have specific places for each kind of experimentation:<br />
<br/><br/><br />
Rooms dedicated for:<br />
<br/><br />
<ul><br />
<li>Baths</li><br />
<br />
<li> Electrophoresis migration</li><br />
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<li>Incubators</li><br />
<br />
<li>Freezer and refrigerator</li><br />
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<li>Autoclave</li><br/><br />
</ul><br />
We use our personal autoclave in the lab to decontaminate and sterilize our materials.<br />
</div> <br />
</div><br />
<br />
<h2>BioBrick parts safety issues</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
A GMO may contain harmful parts and be dangerous for the environment. The potential danger of a GMO is also due to its potential interactions with the environment. When an iGEM project is conceived, the team has to think about the possible interactions of their parts with the environment and design tests to document the effects of their parts on the environment. Synthetic biology is a science which worries people, so we have to provide them the insurance of the complete safety of our experiments.<br/><br />
<br/><br />
<strong>Our molecules : Lysostaphin, Dispersin, Surfactin</strong><br />
<br/><br/><br />
None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Lysostaphin, an endopeptidase specific to the cell wall peptidoglycan of <i>Staphylococcus</i>, is an extremely potent antistaphylococcal agent. Lysostaphin has been used in animals and topically in human against certain infections. Concerning the safety precautions it is indicated to not breathe gas/fumes/vapor/spray, to avoid contact with skin and eyes.">lysostaphin</span>, <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Surfactin is a lipopeptide antibiotic and is used as a surfactant to mediate flux of mono- and divalent cations, such as calcium, across lipid bilayer membranes. It is a powerful biosurfactant which can cause lysis of erythrocytes and bacteria. It is also known as a clotting inhibitor. However, considering the concentrations in which it is produced by bacterial cultures, the risk is minimal. In addition, the “Biofilm Killer” GMO will produce the molecule only locally, further reducing the risks.">surfactin</span> and <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|DispersinB is commercialized by Kane Biotech. It should be used in conjunction with an antibiotic or an anti-microbial, such as silver. Last year, Kane Biotech provided a brief document on DispersinB wound spray to the FDA, in preparation for the submission of an Investigational New Drug Application. They have started clinical trials in order to test the effect of this molecule on infected people. To date, there is no evidence for a negative effect of this molecule on human health.">dispersin</span>. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here. <br />
<br/><br />
<br/><br />
<strong>Our chassis</strong><br />
<br/><br/><br />
In our Biofilm Killer project, we aim to construct a bacterium able to destroy biofilms and to synthesize biocide molecules or a biosurfactant in order to develop a novel method to remove biofilms and clean surfaces in several industrial processes. So, we chose a strain that would have a minimum impact on the process itself, the quality of the product and biological treatment that could exists on site (waste water treatment plant).<br />
<br/><br/><br />
Our genetic constructions have been transferred into a strain of <i>Bacillus subtilis</i>. This species are well known by researchers but also by many industries such as the food industry. <i>Bacillus subtilis</i> is also widely used in the field as a biocontrol agent against plant pathogens.<br />
<br/><br/><br />
It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA)<br />
<br/><br/><br />
<div style="margin:30px;font-style:italic;">"The <i>Bacillus subtilis</i> species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA)[10] and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1]</div><br />
<br/><br/><br />
But <i>Bacillus subtilis</i> is also known to produce endospores that are resistant to different treatments (heat, UV irradiation…). A possible impact on human health cannot be excluded. Indeed, it has been shown that the ingestion of endospores of non-modified <i>B. subtilis</i> does not prevent their germination into the gastrointestinal tract (GIT) [4]. The bacteria are even capable of growth in the lower part of the GIT. But, in this particular case, the consequence seems to be beneficial on health. The presence of <i>B. subtilis</i> in the GIT seems to be linked to the development of the gut-associated lymphoid tissue (GALT). This is one of the reason <i>B. subtilis</i> can be used as a probiotic.<br />
<br/><br/><br />
When the environment becomes unadapted to its growth, the bacterium induces the formation of an endospore in its intracellular compartment [2]. The core of the endospore contains the bacterial chromosome. The core is protected by a double layer membrane, called the forespore. This membrane is itself protected by the coat, composed by multilayered proteins. In <i>B. subtilis</i>, the Cot family proteins is widely involved in the formation of the coat. The resistance of the spore is dependant on the state of hydratation of the core. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism. In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.<br />
<br/><br/><br />
Sporulation can be hazardous to the environment and humans because spores are resistant to most cleaning procedure and thus can survive for a very long time, even in extreme conditions [3]. Spores can be dispersed in the environment and turn into germination process leading to the contamination of new areas previously devoid of this micro-organism.<br />
<br/><br/><br />
In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.<br />
<br/><br/><br />
The high spore resistance to adverse conditions imply that avoiding the induction of sporulation is one task that we need to consider in our project if we use a sporulant <i>B. subtilis</i>.<br />
<br/><br/><br />
If the cells were released in the environment after the germination of spores, we estimate the chance of survival to be minimal, because strain 168 has been modified and habituated to laboratory conditions. Even so, as mentioned below, <i>B. subtilis</i> is not a pathogenic bacterium.<br />
<br/><br/><br />
<strong>For all these reasons, and in the aim of an industrial application, we propose to introduce our genetic constructions in an NON-sporulant <i>B. subtilis</i> strain, such as QB1133.</strong><br />
<br/><br/><br />
The use of a non sporulant <i>B. subtilis</i> to host our genetic construction would not significantly impact neither the environment nor the human health. The odds of the bacteria surviving to the low pH of the stomach, and the very hard life conditions (anaerobia, pH, competition with the intestinal flora) are very low in the vegetative state.<br />
<br/><br/><br />
In addition to the use of non sporulant mutant of <i>B. subtilis</i>, we could enhance safety by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"<br />
<br/><br/><br/><br />
<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br/><br />
1 European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.<br />
<br/><br />
2 Morphogenesis of <i>Bacillus</i> Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)<br />
<br/><br />
3 Role of the Spore Coat Layers in <i>Bacillus subtilis</i> Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626<br />
<br/><br />
4 The Intestinal Life Cycle of <i>Bacillus subtilis</i> and Close Relatives, Duc, Tran T. Hoa, Claudia R. Serra, Adriano O. Henriques Nguyen K. M. Tam, Nguyen Q. Uyen, Huynh A. Hong, Le H.and Simon M. Cutting, J. Bacteriol. 2006, 188(7)<br />
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<br />
<h2>Biosafety group</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
Our institution (INSA Lyon) has a biosafety group. However, we have a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students follow a 4 hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training all along the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies to all the french biosafety regulations.<br />
<br/><br/><br />
As far as the legal aspect is concerned, these is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is quite restrictive, based on the precautionary principle. Even though synthetic biology doesn’t yet have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.<br />
</div><br />
</div><br />
<br />
<h2>New ideas for safety in iGEM</h2><br />
<div class="wrapper"><br />
<div class="contenuTexte"><br />
After standard parts, why not a standard chassis ?<br />
<br/><br/><br />
The INSA de Lyon team proposes that a “safety kit” should be provided to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.<br />
<br/><br/><br />
This kit should contain:<br />
<br/><br />
<ul><br />
<li>a collection of chassis that could be used to receive the DNA constructions. The strains’ genome would be the result of the gene knockout method which is used to inactivate a specific gene. With the new genetic technologies that evolve each day, now it is even possible to purchase a commercial Gene Knockout System which can be designed to knock-out the gene of interest. For example, an applications of this genetic instrument is the construction of auxotrophic strains (or nutrient-deficient) which are mutant strains of bacteria that are unable to grow on minimal media. The proliferation of these organisms outside the laboratory is limited because the lab chemicals are not found in nature.</li><br />
<br />
<li>a toxin/antitoxin system coupled with a lysing agent/cytotoxic compount/degradation agent which is provided on a separate backbone (it would leave the choice to the participants whether or not they want to use it). If the strain containing this system is released, horizontal gene transfer between a non-natural bacteria and a natural one is prohibited. Thus, with the anti-toxin inside the chassis’ genome, and the toxin and cytotoxic compound in the plasmid DNA, the horizontal gene transfer will induce the death of the recipient.<br />
</li><br />
<li>a strain suicide option represented by a gene-killer (also found on a backbone) which is activated in the absence/presence of a certain substrate. For example, Contreras <i>et al.</i> (1991) [1], proposed the construction of a confined strain capable of digesting polluted substance. In the absence of pollutant in the environment, a suicide gene is expressed and the cell is destructed.<br />
</li><br />
</ul><br />
<br/><br />
We are aware that engineering these DNA sequences is not easy. This is why we suggest that a new section or a new reward should be proposed : “ Best Safety Device”. For teams it would be a real challenge and their investment for creating new safety devices would be rewarded.<br />
<br/><br/><br />
In synthetic biology the safety and security measures are of primary importance. Every team should have a proper formation in safety and security issues. INSA de Lyon team suggests that one of the competition requirements should be a diploma validating a proper safety formation.<br />
<br/><br/><br />
<br />
<div style="font-size:12px;font-style:italic;"><br />
<strong>References</strong><br />
<br/><br/><br />
1 Contreras A., Molin S, Ramos JL. 1991. Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics. Applied and Environmental Microbiology.<br />
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{{Lyon-INSA/pub}}</div>Suxiaohuihttp://2012.igem.org/Team:Lyon-INSA/teamTeam:Lyon-INSA/team2012-09-27T01:25:18Z<p>Suxiaohui: </p>
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<p> I am a third-year student in Biochemistry and Biotechnologies at INSA Lyon. I am taking part in the iGEM competition for the first time. This project is a good way for me to improve my organization skills, ingenuity and dynamism which will be very useful for the future. </br>Outside of school, I enjoy cooking, playing handball and mainly traveling whenever possible! So I believe on the talent of her team to discover Amsterdam and of course Boston! </p><br />
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Alex Mizgier<br />
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<p>Also known as Hairix, I am a third-year biochemist at INSA Lyon. I am from Chile, specifically from the northern limit of Western Patagonia.<br/><br />
I got into the iGEM project because I think that it’s an unique experience and an excellent opportunity to learn more about bacterial manipulation and synthetic biology.<br/><br />
Apart from microbiology, I like reading, playing rugby, bicycle touring, and everything that involves nature and outdoor activities. You will find in me a knight-errant.<br/></p><br />
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Ioana Sandu<br />
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<p>I am a third-year Biochemistry student. I decided to join the Lyon team because I believe that the iGEM competition is a challenging experience that will put myself to the test. </br>Apart from synthetic biology, I like dancing, star-gazing and reading while listening to music (the older, the better).<p><br />
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Audrey Masi<br />
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<p>Also known as Squirrelix because I used to climb up on trees and hide in narrow cupboards to make jokes to my friends.<br />
I am a third-year bioengineer.<br />
I am joining the IGEM team after a first experience in molecular biology to improve my knowledge and discover the synthetic biology approach.</br><br />
Outside of iGEM, I am interested in rock dance, cooking, traveling, and above all that literature.</p><br />
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Marion Wolfovski<br />
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<p>Also known as Rasberrix Olympix, I have a bachelor's degree in Sciences and I am now studying Biochemistry and Biotechnologies. This is my first participation in the iGEM competition. I love new challenges and iGEM is an amazing opportunity to manage together my passion for biology and building a project : a scientific adventure.</br> I love sports and making cookies for my lovely team !</p><br />
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Marion Traouan<br />
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<p>I am an undergraduate student in biochemistry and biotechnologies at INSA Lyon. This is my first participation in an iGEM team. I consider this as an opportunity not only to acquire specific qualifications very useful for the future, but also to participate in the construction of an attractive project from scratch.<br/><br />
Besides my interest in biosciences, I am into sports like basket-ball or volley-ball, reading and cinema. I hope my "blondness" won't be an obstacle to the realization of this project.</p><br />
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Alexandre Duprey<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/b/ba/AlexD.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/6/64/Alexmizgier.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>This year, I’m back for another iGEM competition. I’m still at INSA Lyon, fourth-year biochemistry and biotechnology, and still interested in how we can regulate parts to make them work together. However this time I’m rather advising on the project with the intent to improve our weak points (who said modelling ?), and making sure the newcomers don’t make (too many) mistakes. </br> Otherwise I play video games. A lot. Too much...</p><br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Béryl Royer-Bertrand<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/5/53/Beryl.JPG" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cd/Beryl_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<p>This is my second participation in the iGEM competition with the INSA Lyon team. In fourth year in Bioinformatics and Modeling, I’m helping this year the team in the modelling part from Scotland, where I am in academic exchange. </p><br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Viviane Chansavang<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/2/23/Viviannechansavang.jpg" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/a/a0/VivianeFun.png" width="180px"/><br />
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<div class="textePres"><br />
<p>iGEM rocks so badly, when you do it once, you just wanna do it again :) </p> <br />
<p>But this year, I'm keeping a low profile, just helping out the newbies to settle in and feel comfy in the lab. I'm still enjoying working around bacteria, apart from <i>B. subtilis</i> producing lysostaphin, because it smells so badly you just wanna die when you happen to be working in the same room!</p><br />
<p>I also like playing volleyball, cooking, baking, eating and above all traveling and meeting new people ^^ See y'all at the Jamboree! </p><br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Clemence Gonthier<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/Clemencegonthier.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>When you participate in iGEM once, you just want to take part in this great adventure again !</p><br />
<br />
<p>In my fourth year of Biochemistry and Biotechnology, I’m still enjoying microbiology, modifying bacteria and creating new plasmids!<br />
I’m this time on the advisor side... Helping the new team members searching for sponsors and guiding them for experiments in the lab.</p><br />
<br />
<p>In my free time, I like dancing, playing the piano and traveling !</p><br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Bastien Doix<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/78/Bastien.JPG" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4e/Bastien_fun.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>I am a third-year student in Biochemistry and Biotechnology at INSA Lyon and taking part in iGEM for the first time. I am joining the iGEM team to have a first sight of what genetics and bacterial manipulation is and to step into something different from studies.</br> I like sports, skiing and computer graphics and hope to make it to Boston.</p><br />
<br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Patricia Gifu<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/Patricia.JPG" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>I am an undergraduate student in Biochemistry and Biotechnology at INSA de Lyon. This year, I participate for the first time in iGEM. I am very confident that INSA Lyon team is going to win because the team members worked very hard on the project and they all are skillful. I am passionate by biosciences. </br>Outside the school I like watching movies and traveling whenever I have time.</p><br />
<br />
</div><br />
</div> <br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Rémi Hocq<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/8/8c/Remy.jpg/774px-Remy.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/1/10/Remy2.JPG/397px-Remy2.JPG" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>I am also known as Clumsix for often being … clumsy. Hell, I am the kind of guy who managed to cut my finger trying to open a bottle of beer and for the record, I was actually using a bottle-opener. My clumsiness is even contagious : Gaël and Yoann also cut their finger during the summer.</br><br />
<br />
Anyway, I am also a third-year bioengineering student who love music (especially hard rock !), good food (though I am skinny and absolutely don't know why) and traveling. I may be a bit of a nerd too as I am a videogames fanatic.</p><br />
</div><br />
</div><br />
<div class="presentation presStud"><br />
<div class="nomPres"><br />
Carine Gimbert<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/d/dc/Carine.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Carine_fun.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>Also known as Stoatix, it is my first participation in the INSA-Lyon team. In my previous studies, I worked on two projects about bioremediation and production of vanillin using biotechnology. So being part of the iGEM team is really important to continue learning how to manage bacteria and working in a student team. </br>I am fond of dance, photography and art in general. I live in a town where the international short film festival takes place : Clermont-Ferrand and one of my favorite is <a href="http://www.youtube.com/watch?v=DdLehwjV4pc">“la révolution des crabes”</a></p><br />
</div><br />
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<div class="presentation presStud"><br />
<div class="nomPres"><br />
Xavier Tholot<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/Xavier.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/81/Xavier_fun.jpg" width="180px"/><br />
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<div class="textePres"><br />
<br/><br />
I am a third-year student in computer sciences at INSA Lyon. <br/><br />
I come from Clermont-Ferrand, the city where Michelin was created, which isn't far from Lyon.<br/><br />
I am fond of music (I play the guitar and the clarinet), and also of computers : this year, I joined the Lyon-INSA team to help them improving their wiki, and make it as great as possible. <br />
</div><br />
</div><br />
</div><br />
<h2>Advisors</h2><br />
<div class="wrapper"><br />
<div id="advisorListing"><br />
<div class="advisorName"><br />
Gaël Chambonnier<br />
</div><br />
<div class="advisorName"><br />
Fanny Springer<br />
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<div class="advisorName"><br />
Philippe Thomas<br />
</div><br />
<div class="advisorName"><br />
Romain Briandet<br />
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</div><br />
<br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Gaël Chambonnier<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d8/GaelChambo.jpg" width="180px"/><br />
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<div class="textePres"><br />
Hi everybody, here is Chambix!! I just graduated from the INSA Lyon in Biotechnologies but after two participations at the iGEM Competition, I couldn’t do anything but continue my studies with a master in bioinformatics.<br />
After two years working in the lab for the team, I came back this year trying to help out the new team, sharing my experiences. Actually, they didn’t really need my help. They’ve done a great job with Bacillix. And for sure you’ll have to deal with them in Amsterdam and Boston. <br />
</div><br />
</div><br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Fanny Springer<br />
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<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
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<img src="https://static.igem.org/mediawiki/2012/1/12/Fanny.JPG" width="180px"/><br />
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<div class="textePres"><br />
I've joined the INSA-Lyon team this year to see how we can change the world using genetically engineered material !<br />
</div><br />
</div><br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Philippe Thomas<br />
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<div class="photoPres"><br />
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<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/9/99/Photo_Philippe.jpeg" width="180px"/><br />
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<div class="textePres"><br />
<p>This year, Asterix and his friends have travelled until Buenos Aires in Argentina... They meet some strange people living riding horses, drinking mate, eating very good meat and raising cows in the infinite pampa! I’m one of this gauchos! I‘m currently working in Buenos Aires for 18 months as an Engineer in biotechnology in Sanofi Pasteur. As it is not so easy to assist meeting and perform some experiments, the best for me was to be an advisor!</br><br />
I try to help out my Gallic friends when some extra work is needed and give my best as advisor, sharing my experiences of the iGEM competition with them! El hombre de la pampa can’t wait to see the Lyon Biosciences Team rockin’ Amsterdam and Boston with our very powerfull bacteria! </p> <br />
<br />
</div><br />
</div><br />
<div class="presentation adPres"><br />
<div class="nomPres"><br />
Romain Briandet<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e4/Photo_romain_briandet.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>I am the leader of the biofilm group at the INRA Micalis Institute. I have focused my research on microbial biofilms present in the food chain with special emphasis on their role in the persistence of pathogens. One of my main scientific line of interest is to identify the link between the 3D spatial organization of biofilms and the survival mechanisms of cells in face to the exposition of antimicrobials. Recently, my team reported that a tiny proportion of certain <i>Bacillus</i> species can tunnel through biofilms, creating pores that allow molecules to flow in. We are now evaluating the industrial benefit to pretreat target biofilms with swimming <i>Bacilli</i> cocktails to increase their efficacy and reduced their ecological impact.</p><br />
</div><br />
</div><br />
</div><br />
<h2>Instructors</h2><br />
<div class="wrapper"><br />
<br />
<div id="instructorListing"><br />
<div class="instructorName"><br />
Corinne Dorel<br />
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<div class="instructorName"><br />
Valérie Desjardin<br />
</div><br />
<div class="instructorName"><br />
Agnès Rodrigue<br />
</div><br />
<div class="instructorName"><br />
Yoann Louis<br />
</div><br />
<div class="instructorName"><br />
Philippe Oger<br />
</div><br />
<div class="instructorName"><br />
Olivier Brette<br />
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</div><br />
<br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Corinne Dorel<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/7/77/CorinneDorel.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/0/01/145092_SHKN6I8LSIZCUWCZ4FW1O2JUKJVR7Q_abraracourcix_H113219_L.jpg" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>My teaching activities in the Microbial Genetics mainly take place within the Biosciences department at INSA Lyon, but also at the Ecole Normale Supérieur de Lyon and at the University Claude Bernard Lyon 1.<br/><br/><br />
<br />
My research work is based on the understanding of genetic mechanisms involved in the formation of biofilms and the contamination of materials. Being the Communications officer for the Biosciences department, my participation in iGEM 2012 is part of a strategy to promote and share knowledge in the field of research in genetic engineering and more generally in Biological Sciences.</p><br />
</div><br />
</div><br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Valérie Desjardin<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Valerie.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1b/G13b.gif" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
Associate professor at the Institut National des Sciences Appliquées de Lyon<br/><br/><br />
<br />
<p>After a Master of Advanced Studies in Biochemistry and a PhD in Chemistry and Science and Techniques of waste, now I teach Chemistry classes and also a class dealing with radioactive waste management in the Energy Engineering and Environment department. <br/> My research work at the Laboratory of Civil engineering and Environmental engineering (LGCIE) is currently aimed at the study of biophysicochemical interactions of pollutants in various compounds (soils, sediments, municipal solid waste) using molecular biology tools. I am very excited to take part in the iGEM 2012 project which leads to the development of a synergy, already launched, between the Biosciences department and Environmental sciences.</p><br />
</div><br />
</div><br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Agnès Rodrigue<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/Agnesrodrigue.jpg" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/2/2f/Falbala.gif" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>As an associate professor I find that iGEM is an unique experience, a place to mix teaching and practice in a long term project relying on students’ motivation. My teaching interests are microbiology, molecular biology, protein engineering and bioinformatics. <br/> My research topic focus on understanding the molecular mechanisms involved in bacterial adaptation to a metal-rich environment, from molecular interactions to population biology. Apart from the basic approaches, this subject offers also the opportunity to develop biological tools for the bioremediation of spoiled environment or the in situ detection of toxic compounds for instance, developments which I’m interested in. My motivation for iGEM is the same: conception and application. </p><br />
</div><br />
</div><br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Yoann Louis<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Teteyoann.jpg" width="180px"/><br />
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<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9f/Yoyoy.jpg" width="180px"/><br />
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</div><br />
<div class="textePres"><br />
<p>Young ;) ) Associate professor at the Institut National des Sciences Appliquées de Lyon</p><br />
<br />
<p>Up to now my work is focused on the trace metal behavior in the environment and their interaction with dissolved organic matter in aquatic ecosystems.</p><br />
<br />
<p>Now my work at the LGCIE laboratory is mainly <strike>to develop a magic potion to be stronger than Asterix </strike>to study trace metals/ organic matter behavior in various waste and to give an expertise on their potential toxicity depending on the bio-physico-chemical conditions of the studied site.<br />
As a chemist, my interest in the iGEM project is to have a working approach angle allowing the improvements of our environment thanks to our complementarity.</p><br />
</div><br />
</div><br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Philippe Oger<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Tetephiloger.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/8/82/G15b.gif" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Also known as Piezophilix because I work on high-pressure adaptation in microorganisms.<br />
My research is focussed on adaptation mechanisms in microbes from the deep-biosphere, and mainly in Archaea isolated from deep-sea hydrothermal vent systems.<br />
Our aim is to identify and quantify what genetic modifications make our favorite model, <i>Pyrococcus yanaosii</i>, require 500 times the atmospheric pressure for growth, when everybody else's favorite lab rat, <i>E. coli</i>, cannot even growth at the same hydrostatic pressure.<br />
My teaching activities at the University of Lyon deal with petroleum reservoir microbiology and the use of biosignatures for the study of past and present environments.</p><br />
<br />
</div><br />
</div><br />
<div class="presentation insPres"><br />
<div class="nomPres"><br />
Olivier Brette<br />
</div><br />
<div class="photoPres"><br />
<div class="photoNormale"><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/Obrette.png" width="180px"/><br />
</div><br />
<div class="photoCool"><br />
<img src="https://static.igem.org/mediawiki/2012/d/db/G30b.gif" width="180px"/><br />
</div><br />
</div><br />
<div class="textePres"><br />
<p>Associate Professor in economics at the Institut National des Sciences Appliquées (INSA) de Lyon</p><br />
<br />
<p>I teach the "economics of firm", "innovation economics", as well as the "economics of globalization" to engineering students.</p><br />
<br />
<p>I am affiliated with the CNRS Research Unit "Environment, City, Society" (EVS), where I pursue my research activities in theoretical and applied economics. From a theoretical viewpoint, my research work aims at developing the methodological, conceptual, as well as behavioral foundations of Institutional and Evolutionary Economics. I resort to this theoretical framework to deal with different kinds of issues in innovation economics and economic geography.</p><br />
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