http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Tiff2012.igem.org - User contributions [en]2024-03-29T12:26:04ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Evry/AttributionsTeam:Evry/Attributions2012-10-27T03:11:26Z<p>Tiff: </p>
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<h1>Attributions</h1><br />
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<h2>Many thanks to all those who helped us, advised us and encouraged us over the summer !</h2><br />
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<h3>The team</h3><br />
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The team is composed of a mix of students, from L2 (2nd year after baccalaureate) to M2 (5th year after baccalaureate). We worked together from mid june to the end of september, some full time, some part time. More details can be found on the team page.<br />
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<h3>Funding and administrative management</h3><br />
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The team did most of the fundraising work, creating a sponsoring file, contacting potential sponsors and presenting the project. We managed the money ourselves, through Genopole and through a "1901 law" association. Genopole supported our project, paying inscription and jamboree participation fees, as well as donating 5000 euros towards the project. Evry university contributed 7200 euros. Sanofi donated 15 000 euros, and the French embassy in the United States donated 1500 euros. Our other sponsors helped us by providing discounts or free samples. <br />
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<p>Many people at iSSB helped us securing funding and manage the team, notably Jean-Loup Faulon with the Sanofi funding, Dominique Zeliszewski with the sponsoring file preparation and team organisation, Joan Hérisson with setting up the lab, Maelle Cochennec who managed the money we kept at Genopole. Our work would also not have been possible without the help of Sylvie Bobelet and Bernadette Lauret, who solved so many problems the team encountered over the summer. Our supervisor Thomas Landrain was very important to the success of the team, as he brought several important members to the team, he advised us with Nicolas Pollet a lot on the strategic design of our scientific project and helped organizing the human practices in coordination with La Paillasse. Our supervisor Alfonso Jaramillo encouraged us to launch the team and opened his lab to the team.</p><br />
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<h3>Cloning</h3><br />
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All cloning work was done by the team in <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a>, Evry. Supervisors Thomas Landrain and Andrew Tolonen gave advice on cloning strategy throughout the project. <br />
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<h3>Animal work</h3><br />
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Adult frogs are kept in the animal house shared between the Metamorphosys team of iSSB and Watchfrog (a company). The adult frogs were handled by Aurore Thelie from Nicolas Pollet's team (for HCG injection, egg and sperm recovery). <br />
For the auxin toxicity tests, auxin penetration experiments and the <i>E. coli </i> to <i> Xenopus </i> communication devices, the fertilised eggs were provided by the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> team of the iSSB. <br />
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For all tests of biobricks in <i>Xenopus </i>, the team was provided with unfertilised eggs and sperm, and <i>in vitro </i> fertilisation and DNA injection were done by team members under the supervision of Nicolas Pollet. Eggs were sorted and kept and analysed by the team, in the Metamorphosys labs.<br />
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<h3>Microscopy</h3><br />
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Most microscopy photos were taken in the Metamorphosys labs by the team, with the help and supervision of Nicolas Pollet, Aurore Thélie and Léna Vouillot (Postdoc and PhD student in Metamorphosys team).<br />
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We would like to thank Dr. Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for letting us use LSM 510 META Laser Scanning Microscope from Zeiss, which was used with his help and that of Nicolas Pollet and Aurore Thélie for high definition fluorescence images. <br />
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<h3>HPLC</h3><br />
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Many thanks to Damien Baud (Genoscope, Evry), who run our samples on HPLC, and to our advisor Anna who helped us analyse the spectra. <br />
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<h3>Mass spectrometry</h3><br />
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Our beautiful advisor Anna Mlynarczyk ran our samples on LTQ-Orbitrap-XL Mass spectrometer in the LAMBE laboratory (Evry University). Thank you to Veronique Legros for advising and help using it. <br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T03:44:38Z<p>Tiff: </p>
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<h1>Project overview</h1><br />
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For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
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<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
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<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
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<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
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<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
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<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
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<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
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<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
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In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
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<a href="https://2012.igem.org/Team:Evry/AIDSystem"><center>More details here...</center></a><br />
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<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
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Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
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<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
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Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
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<a href="https://2012.igem.org/Team:Evry/Modeling"><center>More details here...</center></a><br />
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<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
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Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
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<a href="https://2012.igem.org/Team:Evry/HumanPractice"><center>More details here...</center></a><br />
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<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-27T01:22:11Z<p>Tiff: </p>
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<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
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<h2>Establishment of a new chassis</b></h2><br/><br />
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<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
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<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
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<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
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<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
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<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
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<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
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<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
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<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
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<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
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To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
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So far we have made 3 new plasmid backbones with different promoters :<br />
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<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
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Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
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<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
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<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
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<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
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<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
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<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-27T01:21:55Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" alt="Image unavailable" width="100px" /></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
</p><br/><br />
<br />
<br />
<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
<p><br />
<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
<br/><br/></p><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br />
<br />
<ul><br />
<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
<br/><br />
To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
<br/><br />
<br/><br />
So far we have made 3 new plasmid backbones with different promoters :<br />
<br/><br/><br />
<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
<br/><br />
<br />
Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
</ul><br />
<br />
<br />
<br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-27T01:21:16Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" alt="Image unavailable" width="100px" /></center><br />
<br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
</p><br/><br />
<br />
<br />
<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
<p><br />
<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
<br/><br/></p><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br />
<br />
<ul><br />
<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
<br/><br />
To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
<br/><br />
<br/><br />
So far we have made 3 new plasmid backbones with different promoters :<br />
<br/><br/><br />
<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
<br/><br />
<br />
Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
</ul><br />
<br />
<br />
<br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-27T01:21:01Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" alt="Image unavailable" width="50px" /></center><br />
<br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
</p><br/><br />
<br />
<br />
<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
<p><br />
<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
<br/><br/></p><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br />
<br />
<ul><br />
<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
<br/><br />
To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
<br/><br />
<br/><br />
So far we have made 3 new plasmid backbones with different promoters :<br />
<br/><br/><br />
<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
<br/><br />
<br />
Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
</ul><br />
<br />
<br />
<br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-27T01:20:46Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" alt="Image unavailable" width="950px" /></center><br />
<br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
</p><br/><br />
<br />
<br />
<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
<p><br />
<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
<br/><br/></p><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br />
<br />
<ul><br />
<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
<br/><br />
To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
<br/><br />
<br/><br />
So far we have made 3 new plasmid backbones with different promoters :<br />
<br/><br/><br />
<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
<br/><br />
<br />
Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
</ul><br />
<br />
<br />
<br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T01:03:26Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/e/e2/Hormone.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/a/a4/GoldeN.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/GB"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/5/5b/ModelingDHIUHEIUHD.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/Modeling"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/ba/HumanPractice.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br><br><br />
<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
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<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T00:55:13Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/e/e2/Hormone.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://static.igem.org/mediawiki/2012/a/a4/GoldeN.png"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://static.igem.org/mediawiki/2012/5/5b/ModelingDHIUHEIUHD.png"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://static.igem.org/mediawiki/2012/b/ba/HumanPractice.png"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
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<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T00:53:05Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/e/e2/Hormone.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 300px; height: 50px; text-align: center; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
!--><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T00:51:57Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/b/bd/Xenope.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
!--><br />
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<li id="disclaimer"><a href="/2011.igem.org:General_disclaimer" title="2011.igem.org:General disclaimer">Disclaimers</a></li><br />
</ul><br />
<br />
</div> <!-- close footer --><br />
</div> <!-- close footer-wrapper --><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/main-img.jsTeam:Evry/main-img.js2012-09-27T00:27:33Z<p>Tiff: </p>
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level3benchwork:"#FFBF3E",<br />
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}<br />
/*Thibault BEGIN*/<br />
var theUI = {<br />
<br />
// ['The French Froggies Project!','Xenopus as a chassis','Hormonal Communicati','Modelingxxxxxxxxxxxx','GoldenBricksxxxxxxxx', 'Human Practicexxxxxx', 'The Teamxxxxxxxxxxxx']<br />
<br />
nodes:{"The French Froggies Project!":{color:"#51C215", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/Project', url:'https://static.igem.org/mediawiki/2012/c/cf/French.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
<br />
"Xenopus as a chassis":{color:"#63a358", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/FrenchFrog', url:'https://static.igem.org/mediawiki/2012/b/bd/Xenope.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
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<br />
"Hormonal Communicati":{color:"#852c2b", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/AIDSystem', url:'https://static.igem.org/mediawiki/2012/e/e2/Hormone.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Auxin emitter":{color:"#852c2b", alpha:0, link:'AIDSystem#auxin'},<br />
"Auxin receiver":{color:"#852c2b", alpha:0, link:'AIDSystem#AID'},<br />
"Auxin toxicity":{color:"#852c2b", alpha:0, link:'AuxinTOX'},<br />
"Auxin uptake":{color:"#852c2b", alpha:0, link:'auxin_uptake'},<br />
<br />
"Modelingxxxxxxxxxxxx":{color:"#1A5291", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/Modeling', url:'https://static.igem.org/mediawiki/2012/5/5b/ModelingDHIUHEIUHD.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"General Model":{color:CLR.level3model, alpha:0, link:'ODE_model'},<br />
"ODEs derivations":{color:CLR.level3model, alpha:0, link:'auxin_pde'},<br />
"Diffusion":{color:CLR.level3model, alpha:0, link:'Auxin_diffusion'},<br />
"Auxin production":{color:CLR.level3model, alpha:0, link:'auxin_production'},<br />
"Auxin reception":{color:CLR.level3model, alpha:0, link:'auxin_detection'},<br />
"Plasmid diffusion":{color:CLR.level3model, alpha:0, link:'plasmid_splitting'},<br />
"Model integration":{color:CLR.level3model, alpha:0, link:'model_integration'},<br />
<br />
"GoldenBricksxxxxxxxx":{color:"#1A5291", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/GB', url:'https://static.igem.org/mediawiki/2012/a/a4/GoldeN.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
<br />
"Human Practicexxxxxx":{color:"#a6953f", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/HumanPractice', url:'https://static.igem.org/mediawiki/2012/b/ba/HumanPractice.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Hi Xenope!":{color:"#a6953f", alpha:0, link:'Team:Evry/HumanPractice/Introduction'},<br />
"Be a chassis?":{color:"#a6953f", alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/modelorganism'},<br />
"Free the frogs!":{color:"#a6953f", alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/freedthefrogs'},<br />
"Chassis, really?":{color:"#a6953f", alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/chassis'},<br />
"Working with Xenopus?":{color:"#a6953f", alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/future'},<br />
"Legislation":{color:"#a6953f", alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/others'},<br />
<br />
"The Teamxxxxxxxxxxxx":{color:"#632e8c", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/Team', url:'https://static.igem.org/mediawiki/2012/9/93/TeamEVRY.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Our Team":{color:"#632e8c", alpha:0, link:'Team'},<br />
"Our Sponsors":{color:"#632e8c", alpha:0, link:'Sponsors'},<br />
"Attributions":{color:"#632e8c", alpha:0, link:'Attributions'},<br />
"Official profile":{color:"#632e8c", alpha:0, link:'https://igem.org/Team.cgi?year=2012'},<br />
"Collaborations":{color:"#632e8c", alpha:0, link:'Collaboration'}<br />
},<br />
<br />
edges:{<br />
<br />
<br />
"The French Froggies Project!":{<br />
"Xenopus as a chassis":{"length":0.8,"weight":2},<br />
"Hormonal Communicati":{"length":0.8,"weight":2},<br />
"Modelingxxxxxxxxxxxx":{"length":0.8,"weight":2},<br />
"GoldenBricksxxxxxxxx":{"length":1.5,"weight":2},<br />
"Human Practicexxxxxx":{"length":0.75,"weight":2},<br />
"The Teamxxxxxxxxxxxx":{"length":0.5,"weight":2}<br />
},<br />
<br />
<br />
"Xenopus as a chassis":{<br />
"Creation of new Xenopus plasmids":{"length":0.75,"weight":1},<br />
"How to micro inject in Xenopus eggs":{"length":0.75,"weight":2},<br />
"Development stages":{"length":0.75,"weight":2},<br />
"Characterization of plasmids and reporters":{"length":0.75,"weight":2}<br />
},<br />
"Hormonal Communicati":{<br />
"The auxin emitter":{"length":1,"weight":2},<br />
"The auxin receiver":{"length":1,"weight":2},<br />
"Auxin toxicity":{"length":1,"weight":2},<br />
"Auxin Uptake":{"length":1,"weight":2}<br />
<br />
},<br />
<br />
"Modelingxxxxxxxxxxxx":{<br />
"General Model":{"length":0.5,"weight":2},<br />
"Rigorous derivations of ODEs":{"length":0.5,"weight":2},<br />
"Diffusion in a realistic geometry":{"length":0.75,"weight":2},<br />
"Auxin production":{"length":0.5,"weight":2},<br />
"Auxin reception":{"length":0.5,"weight":2},<br />
"Plasmid diffusion":{"length":0.5,"weight":2},<br />
"Model integration":{"length":0.5,"weight":2}<br />
<br />
},<br />
<br />
"GoldenBricksxxxxxxxx":{},<br />
<br />
<br />
"Human Practicexxxxxx":{<br />
"Hi Xenope!":{"length":0.5,"weight":2},<br />
"Would you be my chassis?":{"length":0.5,"weight":2},<br />
"Free the frogs!":{"length":0.5,"weight":2},<br />
"I'm a chassis, really?":{"length":0.5,"weight":2},<br />
"Should we work with Xenopus again?":{"length":0.5,"weight":2},<br />
"What the laws says...":{"length":0.5,"weight":2}<br />
},<br />
<br />
"The Teamxxxxxxxxxxxx":{<br />
"Our Team":{"length":0.5,"weight":2},<br />
"Our Sponsors":{"length":0.5,"weight":2},<br />
"Attributions":{"length":0.5,"weight":2},<br />
"Official profile":{"length":0.5,"weight":2},<br />
"Collaborations":{"length":0.5,"weight":2}<br />
}<br />
}<br />
}<br />
/*Thibault END*/<br />
<br />
var sys = arbor.ParticleSystem()<br />
sys.parameters({stiffness:900, repulsion:50, gravity:true, dt:0.015})<br />
sys.renderer = Renderer("#sitemap")<br />
sys.graft(theUI)<br />
<br />
var nav = Nav("#nav")<br />
$(sys.renderer).bind('navigate', nav.navigate)<br />
$(nav).bind('mode', sys.renderer.switchMode)<br />
nav.init()<br />
})<br />
})(this.jQuery)</div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-27T00:18:39Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 300px; height: 50px; text-align: center; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
!--><br />
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title="List of all wiki pages that link here [j]" accesskey="j">What links here</a></li><br />
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title="Printable version of this page [p]" accesskey="p">Printable version</a><br />
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title="Permanent link to this revision of the page">Permanent link</a><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/css_v1Team:Evry/css v12012-09-27T00:18:20Z<p>Tiff: </p>
<hr />
<div>body<br />
{<br />
width: auto;<br />
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/*background:#e9e4de;*/<br />
background-image:url(https://static.igem.org/mediawiki/2012/0/06/Embryos.png);<br />
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<br />
/* la barre titre (baniere igem+ menu haut)*/<br />
#top-section<br />
{<br />
height: auto;<br />
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}<br />
<br />
/*le conteneur de la banière igem avec un lien vers igem*/<br />
#p-logo<br />
{<br />
display:none;<br />
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}<br />
<br />
/* la barre de menu du haut */<br />
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{<br />
color:#000000;<br />
}<br />
<br />
/*les lien de la barre de menu du haut */<br />
#menubar a<br />
{<br />
text-decoration:none;<br />
color:#000000;<br />
}<br />
<br />
/* le menu de haut gauche */<br />
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background-color:transparent;<br />
/*display:none;*/<br />
}<br />
<br />
/* le menu de login haut droite*/<br />
.right-menu<br />
{<br />
color:transparent;<br />
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background-color:none;<br />
right: 15px;<br />
}<br />
<br />
/* les lien du login */<br />
.right-menu a<br />
{<br />
color:transparent;<br />
text-decoration:transparent;<br />
background-color:none;<br />
}<br />
<br />
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{<br />
display:none;<br />
}<br />
<br />
/* le body de la page*/<br />
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}<br />
<br />
/*titre de page */<br />
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/* la boite en bas de page*/<br />
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<br />
/* le cadre sous le body useless */<br />
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<br />
/* ----------------End of the wiki hack -------------------------------------*/<br />
<br />
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<br />
/* Position of the banner */ <br />
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<br />
<br />
/*white font over the grey one on the top*/<br />
#white_thingy<br />
{<br />
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/* notre page !*/<br />
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<br />
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text-align:justify;<br />
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<br />
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/*border:1px solid black;*/<br />
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text-align:justify;<br />
top:210px;/*178px*/<br />
width:940px;<br />
heigth:auto;<br />
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<br />
#menu_evry2012 a<br />
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/*height:18px;*/<br />
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<br />
#menu_evry2012 .menu_list_item_evry2012 ul a<br />
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border: 1px solid #e6e1db;<br />
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<br />
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{<br />
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}<br />
<br />
#menu_evry2012 a:active<br />
{<br />
background-color:#e6e1db;<br />
display:block;<br />
}<br />
<br />
<br />
<br />
<br />
/* Style of the menu level1 */<br />
#menu_list_evry2012<br />
{<br />
list-style:none; <br />
text-align:center;<br />
margin-left:0px;<br />
margin-top:0px;<br />
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}<br />
<br />
.menu_list_item_evry2012<br />
{<br />
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<br />
/*Second level menu*/<br />
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{<br />
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}<br />
<br />
/* 2nd level drop-down box */<br />
.menu_list_item_evry2012:hover ul,<br />
.menu_list_item_evry2012 a:hover ul<br />
{<br />
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.ania<br />
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<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 300px; height: 50px; text-align: center; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
!--><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/css_v1Team:Evry/css v12012-09-27T00:10:03Z<p>Tiff: </p>
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}<br />
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text-decoration:underline<br />
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.ania<br />
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color:#cb6228;<br />
}</div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-26T23:44:11Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" class="moredetails" style="position: relative; margin-left: auto; margin-right: auto; width: 400px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
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<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-26T23:43:15Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 400px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br />
<table style="background:transparent";><br />
<tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><br />
<h3 style="text-align:center; padding-left:20px;"></u><br />
<br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog"><center>More details here...</center></a><br />
<br />
</u></h3></td><br />
</tr></table><br />
<br /><br />
</font><br />
</div><br />
<br />
<br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-26T23:41:59Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot ,us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<br />
<br><br />
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<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
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<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
Merging the power of the Golden Gate multi-fragment cloning technique with the standardization of the Biobrick format. As a result, our team is developing this year a new parts format: the GoldenBricks. This is a new ultrafast cloning technique designed to assemble entire cassette in one shot. Ultimately, the GoldenBricks method is paving the way for future PartsRegistry formats!<br />
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<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-26T23:07:06Z<p>Tiff: </p>
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<div>{{:Team:Evry/template_v1}}<br />
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<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have met synthetic biology laboratories on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>A multi-tissular systems allows testing protein effect into an animal. The expression/degradation of a protein (a protein fused to GFP in example) can be followed in the organism. <i>Xenopus</i> can be used as a biosensor, Organisation for Economic Co-operation and Development (OECD) plan to validate an assay capable of <a href="http://www.oecd.org/chemicalsafety/testingofchemicals/41620749.pdf">detecting thyroid disruptor using <i>Xenopus</i></a>. With our plasmid it is easy to test in 5 days a promoter or/and a reporter in the new chassis <i>xenopus</i> because it contains a working immune/vascular/neurologic/nephrologic/digestive systems.<br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <br />
<a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system</a>.<br />
</p><br/><br />
<br />
<br />
<a name="plasmid" /><h3>The simple molecular strategy to build eukaryotic plasmid ready to use: </h3></a><br />
<p><br />
<br/><i>Xenopus tropicalis</i> represents a challenge as it is a vertebrate but also because it's a new chassis in the iGEM competition. To make sure we meet iGEM’s expectations on time, we have had to develop a new biobrick plasmid backbone compatible with<i> Xenopus tropicalis</i> (but also others vertebrate and fish). The plasmid is produced in bacteria then purified and injected into <i>Xenopus</i>'s eggs. The plasmid can not replicate in eukaryotic cells. As origins of replication are cryptic in <i>Xenopus</i>, the plasmid does not replicate. Therefore, it is only active in the first two or three weeks of development, as the injected plasmid is passed on randomly to daughter cells. After that, it becomes too diluted. <br />
<br/><br/></p><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br/><br />
<br />
<ul><br />
<li><b>Kozac</b> : The Kozak consensus sequence is essential for the initiation of the translation process in eukaryotes. The sequence is the following (gcc)gccRccAUGG, where upper case letters are highly-conserved bases, and lower case letter can vary</li><br/><br />
<li><b>Promoter </b>: The promoter of a gene is located upstream of this gene and initiate its transcription. The promoter is surrounded by the enzyme restriction sites <b>SalI</b> and <b>HindIII</b> in order to be able to switch more easily to other promoters</li><br/><br />
<li><b>5'UTR and 3’UTR</b> : UTRs are Untranslated Region. The B-globin 5' UTR is located upstream of the coding sequence and is involved in 5'RNA capping. The 3'UTR is located downstream of the coding sequence and may contain sequences for the regulation of translation efficiency, mRNA stability, and polyadenylation signals</li><br/><br />
<li><b>SV40 PolyA signal</b> : The simian virus 40 polyadenylation signal is involved in the maturation of the mRNA for translation and is composed of a succession of adenine bases </li><br/><br />
<li><b>Antibiotic resistance genes</b> : This sequence is necessary for the selection of transformed bacteria exposed to the antibiotic</li><br/><br />
<li><b>Biobrick prefix and suffix</b></li><br/><br />
<li><b>Origin of replication</b> : This origin of replication is bacterial, this sequence initiates the replication of DNA</li><br/><br />
By putting all the parts necessary for expression in eukaryotes, we have made plasmids where any coding biobrick (containing Kozak sequence) can be cloned in directly without having to transfer each parts individually. These plasmids can be used to rapidly test genetic constructions in<i> Xenopus tropicalis</i> after a single cloning.<br/> It is possible to easily change promoter with the restriction sites SalI and HindIII. <br/><br />
The plasmid also contains tools to calibrate the system in combination with a model. For instance, it contains sites for in vitro transcription (sp6 sites) of genes to make RNA that can then be injected directly in the embryo, allowing a much finer control of the ratio between levels of different genes during construct testing. <br/><br />
<br/><br />
To test it, inject 2.3 nL of 100 ng.uL-1 plasmid solution into the one cell embryo following the <a href="https://2012.igem.org/Team:Evry/InjectionTuto" target="_blank">injecting tutorial</a>.For a plasmid of 4kb it represents approximately 45 million of plasmids per injection. As the cell divides, plasmids are shared between cells but not replicated so a high concentration of DNA is necessary to ensure there will be DNA in most of the organism. Once the transcription machinery turns on during the development, plasmids are transcribed and translated. Since the tadpole stage starts after a few days, we can work on a whole vertebrate with most organs formed within a week. With different tissues it is possible to diversify the type of expression with different promoters.<br />
<br/><br />
<br/><br />
So far we have made 3 new plasmid backbones with different promoters :<br />
<br/><br/><br />
<li>With a <b>CMV promoter</b> for an ubiquitous expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812000" target="_blank">BBa_K812000</a>), <br />
<li>With a <b>Hsp70 promoter</b> for an inducible expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812300" target="_blank">BBa_K812300</a>),<br />
<li>With a <b>Elastase promoter</b> for a tissue specific expression :<br />
(<a href="http://partsregistry.org/Part:BBa_K812200" target="_blank">BBa_K812200</a>). </p></li><br />
<br/><br />
<br />
Our goal was to provide tools which would allow to rapidly build and characterize constructs in the embryos (along with a synthetic hormone to make them communicate), with tools for debugging (by injecting mRNA) and with ubiquitous, inducible and tissue specific promoters.<br />
</ul><br />
<br />
<br />
<br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in <i>Xenopus</i></h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>pCS2+ GFP-aid: this plasmid contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
</b></b><br />
<p><br />
GFP-aid expression from the embryo at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 20 (one day after injection )to a tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> ~46 (four days after injection). For this tadpole the expression is localized in the skin.</b></b></b><br />
</p><br />
<br />
We characterize the promoter CMV and elastase, not yet for the inducible promoter HSP70.<br />
Reporters characterized: sfGFP, mCitrine, mCFP and GFP-aid.<br><br><br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>This new BioBricked plasmid is ready to use in <i>Xenopus tropicalis</i>. As you can see above the plasmid pCS2+ with the CMV pomoter express the fluorescent protein GFP-aid, but this plasmid carry on important things to express a gene into eukaryotic calls. The 5'UTR region and the 3' UTR region was needed for gene expression in euklaryotic organism. The simplest way to do that was to use a plasmid already used in eukaryotic cells, vertebrate and especially <i>Xenopus tropicalis</i> as pCS2+. This pCS2+ was Biobricked to be compatible with the registry. <br><br />
<br />
The pCS2+ plasmid was characterized and different reporters were expressed under the control of different promoters: <a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a>.<br><br />
<br />
<p><br />
Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Within tadpole, fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.<br />
</p><br />
<br />
<p><br />
Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. Another reason could be that the metabolism of each differentiated cell is different and changes during the tadpole's development.<br />
</p><br />
<br />
<p><br />
Our experiment showed us that plasmids injected do not diffuse in the whole organism. To express a protein with a promoter tissue specific it is a problem, because of the very low the probability to have the plasmid into the expected tissue. To solve the problem it is possible to easily integrate promoters and genes into the <i>Xenopus</i>'s chromosome with the REMI technic [3]. A new plasmid pCS2+ with I-SceI sites upstream the promoter and downstream of the reporter could allow the integration of the sequence between this two I-SceI restriction sites. This plasmid could be useful for the eukaryotic community, they could change promoters easily with SalI and HindIII and the reporter is compatible with the registry (with BB prefix and suffix), and they would have the choice to test a construction by plasmid injection or DNA chromosome integration.<br />
</p><br />
<br />
<p><br />
Moreover, the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell. Integration into the chromosome could prevent it. <br />
</p><br />
<br />
<p><br />
Our project raised important ethics question because the team use tadpole, an animal. We reflect that working with tadpole involved new questions about the animal pain but also about using animals in iGEM and in Synthetic Biology. It made us think about these questions, our reflection is in <a href="https://2012.igem.org/Team:Evry/HumanPractice">Human Practice</a>.</p><br><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in <i>Xenopus</i> tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>REMI (Restriction Enzyme Mediated Integration) and its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants</li> Kahmann R., Basse C., European Journal of Plant Pathology</li><br />
</ol><br />
</div><br />
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_initMouseHandling:function(){<br />
// no-nonsense drag and drop (thanks springy.js)<br />
selected = null;<br />
nearest = null;<br />
var dragged = null;<br />
var oldmass = 1<br />
<br />
var _section = null<br />
<br />
var handler = {<br />
moved:function(e){<br />
var pos = $(canvas).offset();<br />
_mouseP = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
nearest = sys.nearest(_mouseP);<br />
<br />
if (!nearest.node) return false<br />
<br />
// if (nearest.node.data.shape!='dot'){<br />
selected = (nearest.distance < 50) ? nearest : null<br />
if (selected){<br />
dom.addClass('linkable')<br />
window.status = selected.node.data.link//.replace(/^\//,"http://"+window.location.host+"/")//.replace(/^#/,'')<br />
}<br />
else{<br />
dom.removeClass('linkable')<br />
window.status = ''<br />
}<br />
// }else <br />
if ($.inArray(nearest.node.name, ['The French Froggies Project!','Xenopus as a chassis','Hormonal Communication','Modeling','GoldenBricks', 'Human Practice', 'The Team']) >=0 ){<br />
if (nearest.node.name!=_section){<br />
_section = nearest.node.name<br />
that.switchSection(_section)<br />
}<br />
dom.removeClass('linkable')<br />
window.status = ''<br />
}<br />
<br />
return false<br />
},<br />
clicked:function(e){<br />
var pos = $(canvas).offset();<br />
_mouseP = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
nearest = dragged = sys.nearest(_mouseP);<br />
<br />
if (nearest && selected && nearest.node===selected.node){<br />
var link = selected.node.data.link<br />
if (link.match(/^#/)){<br />
$(that).trigger({type:"navigate", path:link.substr(1)})<br />
}else{<br />
window.location = link<br />
}<br />
return false<br />
}<br />
<br />
<br />
if (dragged && dragged.node !== null) dragged.node.fixed = true<br />
<br />
$(canvas).unbind('mousemove', handler.moved);<br />
$(canvas).bind('mousemove', handler.dragged)<br />
$(window).bind('mouseup', handler.dropped)<br />
<br />
return false<br />
},<br />
dragged:function(e){<br />
var old_nearest = nearest && nearest.node._id<br />
var pos = $(canvas).offset();<br />
var s = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
<br />
if (!nearest) return<br />
if (dragged !== null && dragged.node !== null){<br />
var p = sys.fromScreen(s)<br />
dragged.node.p = p<br />
}<br />
<br />
return false<br />
},<br />
<br />
dropped:function(e){<br />
if (dragged===null || dragged.node===undefined) return<br />
if (dragged.node !== null) dragged.node.fixed = false<br />
dragged.node.tempMass = 1000<br />
dragged = null;<br />
// selected = null<br />
$(canvas).unbind('mousemove', handler.dragged)<br />
$(window).unbind('mouseup', handler.dropped)<br />
$(canvas).bind('mousemove', handler.moved);<br />
_mouseP = null<br />
return false<br />
}<br />
<br />
<br />
}<br />
<br />
$(canvas).mousedown(handler.clicked);<br />
$(canvas).mousemove(handler.moved);<br />
<br />
}<br />
}<br />
<br />
return that<br />
}<br />
<br />
<br />
var Nav = function(elt){<br />
var dom = $(elt)<br />
<br />
var _path = null<br />
<br />
var that = {<br />
init:function(){<br />
$(window).bind('popstate',that.navigate)<br />
dom.find('> a').click(that.back)<br />
$('.more').one('click',that.more)<br />
<br />
$('#Model dl:not(.datastructure) dt').click(that.reveal)<br />
that.update()<br />
return that<br />
},<br />
more:function(e){<br />
$(this).removeAttr('href').addClass('less').html('&nbsp;').siblings().fadeIn()<br />
$(this).next('h2').find('a').one('click', that.less)<br />
<br />
return false<br />
},<br />
less:function(e){<br />
var more = $(this).closest('h2').prev('a')<br />
$(this).closest('h2').prev('a')<br />
.nextAll().fadeOut(function(){<br />
$(more).text('creation & use').removeClass('less').attr('href','#')<br />
})<br />
$(this).closest('h2').prev('a').one('click',that.more)<br />
<br />
return false<br />
},<br />
reveal:function(e){<br />
$(this).next('dd').fadeToggle('fast')<br />
return false<br />
},<br />
back:function(){<br />
_path = "/"<br />
if (window.history && window.history.pushState){<br />
window.history.pushState({path:_path}, "", _path);<br />
}<br />
that.update()<br />
return false<br />
},<br />
navigate:function(e){<br />
var oldpath = _path<br />
if (e.type=='navigate'){<br />
_path = e.path<br />
if (window.history && window.history.pushState){<br />
window.history.pushState({path:_path}, "", _path);<br />
}else{<br />
that.update()<br />
}<br />
}else if (e.type=='popstate'){<br />
var state = e.originalEvent.state || {}<br />
_path = state.path || window.location.pathname.replace(/^\//,'')<br />
}<br />
if (_path != oldpath) that.update()<br />
},<br />
update:function(){<br />
var dt = 'slow'<br />
if (_path===null){<br />
// this is the original page load. don't animate anything just jump<br />
// to the proper state<br />
_path = window.location.pathname.replace(/^\//,'')<br />
dt = 0<br />
dom.find('p').css('opacity',0).show().fadeTo('slow',1)<br />
}<br />
<br />
switch (_path){<br />
case '':<br />
case '/':<br />
// dom.find('p').text('a graph visualization library using web workers and jQuery')<br />
dom.find('> a').removeClass('active').attr('href','#')<br />
<br />
$('#Model').fadeTo('fast',0, function(){<br />
$(this).hide()<br />
$(that).trigger({type:'mode', mode:'visible', dt:dt})<br />
})<br />
document.title = "The French Froggies Project!"<br />
break<br />
<br />
}<br />
<br />
}<br />
}<br />
return that<br />
}<br />
<br />
$(document).ready(function(){<br />
var CLR = {<br />
branch:"#b2b19d",<br />
level3benchwork:"#FFBF3E",<br />
level3model:"#4B86C7"<br />
}<br />
/*Thibault BEGIN*/<br />
var theUI = {<br />
<br />
// ['The French Froggies Project!','Xenopus as a chassis','Hormonal Communication','Modeling','GoldenBricks', 'Human Practices', 'The Team']<br />
<br />
nodes:{"The French Froggies Project!":{color:"#51C215", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/Project', url:'https://static.igem.org/mediawiki/2012/1/19/Button1.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
<br />
"Xenopus as a chassis":{color:"#1A5291", shape:"dot", alpha:1, link:'FrenchFrog', url:'ResultsbuttonLowRes.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Creation of new Xenopus plasmids":{color:CLR.level3model, alpha:0, link:'FrenchFrog#plasmid'},<br />
"How to micro inject in Xenopus eggs":{color:CLR.level3model, alpha:0, link:'InjectionTuto'},<br />
"Development stages":{color:CLR.level3model, alpha:0, link:'Stages'},<br />
"Characterization of plasmids and reporters":{color:CLR.level3model, alpha:0, link:'Tadpole_injection1'},<br />
<br />
"Hormonal Communication":{color:"#1A5291", shape:"img", alpha:1, link:'AIDSystem', url:'https://static.igem.org/mediawiki/2012/a/a0/Button6.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"The auxin emmiter":{color:CLR.level3model, alpha:0, link:'AIDSystem#auxin'},<br />
"The auxin receiver":{color:CLR.level3model, alpha:0, link:'AIDSystem#AID'},<br />
"Auxin toxicity":{color:CLR.level3model, alpha:0, link:'AuxinTOX'},<br />
"Auxin Uptake":{color:CLR.level3model, alpha:0, link:'auxin_uptake'},<br />
<br />
"Modeling":{color:"#1A5291", shape:"img", alpha:1, link:'Modeling', url:'https://static.igem.org/mediawiki/2012/5/55/Button5.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"General Model":{color:CLR.level3model, alpha:0, link:'ODE_model'},<br />
"Rigorous derivations of ODEs":{color:CLR.level3model, alpha:0, link:'auxin_pde'},<br />
"Diffusion in a realistic geometry":{color:CLR.level3model, alpha:0, link:'Auxin_diffusion'},<br />
"Auxin production":{color:CLR.level3model, alpha:0, link:'auxin_production'},<br />
"Auxin reception":{color:CLR.level3model, alpha:0, link:'auxin_detection'},<br />
"Plasmid diffusion":{color:CLR.level3model, alpha:0, link:'plasmid_splitting'},<br />
"Model integration":{color:CLR.level3model, alpha:0, link:'model_integration'},<br />
<br />
"GoldenBricks":{color:"#1A5291", shape:"img", alpha:1, link:'GB', url:'https://static.igem.org/mediawiki/2012/f/fd/Button3.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
<br />
"Human Practice":{color:"#1A5291", shape:"img", alpha:1, link:'https://2012.igem.org/Team:Evry/HumanPractice/HumanPractice', url:'https://static.igem.org/mediawiki/2012/8/8f/Button2.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Hi Xenope!":{color:CLR.level3model, alpha:0, link:'Team:Evry/HumanPractice/Introduction'},<br />
"Would you be my chassis?":{color:CLR.level3model, alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/HumanPractice/modelorganism'},<br />
"Freed the frogs!":{color:CLR.level3model, alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/freedthefrogs'},<br />
"I'm a chassis, really?":{color:CLR.level3model, alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/HumanPractice/chassis'},<br />
"Should we work with Xenopus again?":{color:CLR.level3model, alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/HumanPractice/future'},<br />
"What the laws says...":{color:CLR.level3model, alpha:0, link:'https://2012.igem.org/Team:Evry/HumanPractice/HumanPractice/others'},<br />
<br />
"The Team":{color:"#1A5291", shape:"img", alpha:1, link:'Team', url:'https://static.igem.org/mediawiki/2012/5/53/Button4.png'}, //Attribut url pour l'image ajouté /*Thibault*/<br />
"Our Team":{color:CLR.level3model, alpha:0, link:'Team'},<br />
"Our Sponsors":{color:CLR.level3model, alpha:0, link:'Sponsors'},<br />
"Attributions":{color:CLR.level3model, alpha:0, link:'Attributions'},<br />
"Official profile":{color:CLR.level3model, alpha:0, link:'https://igem.org/Team.cgi?year=2012'},<br />
"Collaborations":{color:CLR.level3model, alpha:0, link:'Collaboration'}<br />
},<br />
<br />
edges:{<br />
<br />
<br />
"The French Froggies Project!":{<br />
"Xenopus as a chassis":{"length":0.4,"weight":2},<br />
"Hormonal Communication":{"length":0.6,"weight":2},<br />
"Modeling":{"length":0.4,"weight":2},<br />
"GoldenBricks":{"length":1.5,"weight":2},<br />
"Human Practice":{"length":0.75,"weight":2},<br />
"The Team":{"length":0.5,"weight":2}<br />
},<br />
<br />
<br />
"Xenopus as a chassis":{<br />
"Creation of new Xenopus plasmids":{"length":0.75,"weight":1},<br />
"How to micro inject in Xenopus eggs":{"length":0.75,"weight":2},<br />
"Development stages":{"length":0.75,"weight":2},<br />
"Characterization of plasmids and reporters":{"length":0.75,"weight":2}<br />
},<br />
"Hormonal Communication":{<br />
"The auxin emmiter":{"length":1,"weight":2},<br />
"The auxin receiver":{"length":1,"weight":2},<br />
"Auxin toxicity":{"length":1,"weight":2},<br />
"Auxin Uptake":{"length":1,"weight":2}<br />
<br />
},<br />
<br />
"Modeling":{<br />
"General Model":{"length":0.5,"weight":2},<br />
"Rigorous derivations of ODEs":{"length":0.5,"weight":2},<br />
"Diffusion in a realistic geometry":{"length":0.75,"weight":2},<br />
"Auxin production":{"length":0.5,"weight":2},<br />
"Auxin reception":{"length":0.5,"weight":2},<br />
"Plasmid diffusion":{"length":0.5,"weight":2},<br />
"Model integration":{"length":0.5,"weight":2}<br />
<br />
},<br />
<br />
"GoldenBricks":{},<br />
<br />
<br />
"Human Practice":{<br />
"Hi Xenope!":{"length":0.5,"weight":2},<br />
"Would you be my chassis?":{"length":0.5,"weight":2},<br />
"Freed the frogs!":{"length":0.5,"weight":2},<br />
"I'm a chassis, really?":{"length":0.5,"weight":2},<br />
"Should we work with Xenopus again?":{"length":0.5,"weight":2},<br />
"What the laws says...":{"length":0.5,"weight":2}<br />
},<br />
<br />
"The Team":{<br />
"Our Team":{"length":0.5,"weight":2},<br />
"Our Sponsors":{"length":0.5,"weight":2},<br />
"Attributions":{"length":0.5,"weight":2},<br />
"Official profile":{"length":0.5,"weight":2},<br />
"Collaborations":{"length":0.5,"weight":2}<br />
}<br />
}<br />
}<br />
/*Thibault END*/<br />
<br />
var sys = arbor.ParticleSystem()<br />
sys.parameters({stiffness:900, repulsion:50, gravity:true, dt:0.015})<br />
sys.renderer = Renderer("#sitemap")<br />
sys.graft(theUI)<br />
<br />
var nav = Nav("#nav")<br />
$(sys.renderer).bind('navigate', nav.navigate)<br />
$(nav).bind('mode', sys.renderer.switchMode)<br />
nav.init()<br />
})<br />
})(this.jQuery)</div>Tiffhttp://2012.igem.org/Team:Evry/main-img.jsTeam:Evry/main-img.js2012-09-26T21:31:30Z<p>Tiff: </p>
<hr />
<div>(function($){<br />
<br />
var Renderer = function(elt){<br />
var dom = $(elt)<br />
var canvas = dom.get(0)<br />
var ctx = canvas.getContext("2d");<br />
var gfx = arbor.Graphics(canvas)<br />
var sys = null<br />
<br />
var _vignette = null<br />
var selected = null,<br />
nearest = null,<br />
_mouseP = null;<br />
<br />
<br />
var that = {<br />
init:function(pSystem){<br />
sys = pSystem<br />
sys.screen({size:{width:dom.width(), height:dom.height()},padding:[36,60,36,60]})<br />
<br />
$(window).resize(that.resize)<br />
that.resize()<br />
that._initMouseHandling()<br />
<br />
if (document.referrer.match(/Goldenbricks|Plasmids|AIDSystem/)){<br />
// if we got here by hitting the back button in one of the Benchwork, <br />
// start with the Benchwork section pre-selected<br />
that.switchSection('Benchwork')<br />
}<br />
},<br />
resize:function(){<br />
// canvas.width = .5* $(window).width()<br />
// canvas.height = .5* $(window).height()<br />
sys.screen({size:{width:canvas.width, height:canvas.height}})<br />
_vignette = null<br />
that.redraw()<br />
},<br />
redraw:function(){<br />
gfx.clear()<br />
sys.eachEdge(function(edge, p1, p2){<br />
if (edge.source.data.alpha * edge.target.data.alpha == 0) return<br />
gfx.line(p1, p2, {stroke:"#b2b19d", width:3, alpha:edge.target.data.alpha})<br />
})<br />
sys.eachNode(function(node, pt){<br />
var w = Math.max(20, 20+gfx.textWidth(node.name) )<br />
if (node.data.alpha===0) return<br />
<br />
if (node.data.shape=='img'){<br />
var img_elem = new Image();<br />
img_elem.src = node.data.url;<br />
ctx.drawImage(img_elem, pt.x-w/2, pt.y-w/2, w, w); // Redimensionnement de l'image prévue comme tu le souhaitais !<br />
gfx.oval(pt.x-w/2, pt.y-w/2, w, w, {fill:node.data.color, alpha:0})<br />
}<br />
<br />
else if (node.data.shape=='dot'){<br />
gfx.oval(pt.x-w/2, pt.y-w/2, w, w, {fill:node.data.color, alpha:node.data.alpha})<br />
gfx.text(node.name, pt.x, pt.y+7, {color:"white", align:"center", font:"Arial", weight:"bold", size:12}) <br />
}<br />
<br />
else{<br />
gfx.rect(pt.x-w/2, pt.y-8, w, 20, 4, {fill:node.data.color, alpha:node.data.alpha})<br />
gfx.text(node.name, pt.x, pt.y+9, {color:"white", align:"center", font:"Arial", size:12})<br />
gfx.text(node.name, pt.x, pt.y+9, {color:"white", align:"center", font:"Arial", size:12})<br />
}<br />
})<br />
that._drawVignette()<br />
},<br />
<br />
_drawVignette:function(){<br />
var w = canvas.width<br />
var h = canvas.height<br />
var r = 20<br />
<br />
if (!_vignette){<br />
var top = ctx.createLinearGradient(0,0,0,r)<br />
top.addColorStop(0, "#e0e0e0")<br />
top.addColorStop(.7, "rgba(255,255,255,0)")<br />
<br />
var bot = ctx.createLinearGradient(0,h-r,0,h)<br />
bot.addColorStop(0, "rgba(255,255,255,0)")<br />
bot.addColorStop(1, "white")<br />
<br />
_vignette = {bot:bot}<br />
}<br />
<br />
// top<br />
ctx.fillStyle = _vignette.top<br />
ctx.fillRect(0,0, w,r)<br />
<br />
// bot<br />
ctx.fillStyle = _vignette.bot<br />
ctx.fillRect(0,h-r, w,r)<br />
},<br />
<br />
switchMode:function(e){<br />
if (e.mode=='hidden'){<br />
dom.stop(true).fadeTo(e.dt,0, function(){<br />
if (sys) sys.stop()<br />
$(this).hide()<br />
})<br />
}else if (e.mode=='visible'){<br />
dom.stop(true).css('opacity',0).show().fadeTo(e.dt,1,function(){<br />
that.resize()<br />
})<br />
if (sys) sys.start()<br />
}<br />
},<br />
<br />
switchSection:function(newSection){<br />
var parent = sys.getEdgesFrom(newSection)[0].source<br />
var children = $.map(sys.getEdgesFrom(newSection), function(edge){<br />
return edge.target<br />
})<br />
<br />
sys.eachNode(function(node){<br />
if (node.data.shape=='dot') return // skip all but leafnodes<br />
if (node.data.shape=='img') return // skip all but leafnodes<br />
var nowVisible = ($.inArray(node, children)>=0)<br />
var newAlpha = (nowVisible) ? 1 : 0<br />
var dt = (nowVisible) ? .5 : .5<br />
sys.tweenNode(node, dt, {alpha:newAlpha})<br />
<br />
if (newAlpha==1){<br />
node.p.x = parent.p.x + .05*Math.random() - .025<br />
node.p.y = parent.p.y + .05*Math.random() - .025<br />
node.tempMass = .001<br />
}<br />
})<br />
},<br />
<br />
<br />
_initMouseHandling:function(){<br />
// no-nonsense drag and drop (thanks springy.js)<br />
selected = null;<br />
nearest = null;<br />
var dragged = null;<br />
var oldmass = 1<br />
<br />
var _section = null<br />
<br />
var handler = {<br />
moved:function(e){<br />
var pos = $(canvas).offset();<br />
_mouseP = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
nearest = sys.nearest(_mouseP);<br />
<br />
if (!nearest.node) return false<br />
<br />
// if (nearest.node.data.shape!='dot'){<br />
selected = (nearest.distance < 50) ? nearest : null<br />
if (selected){<br />
dom.addClass('linkable')<br />
window.status = selected.node.data.link//.replace(/^\//,"http://"+window.location.host+"/")//.replace(/^#/,'')<br />
}<br />
else{<br />
dom.removeClass('linkable')<br />
window.status = ''<br />
}<br />
// }else <br />
if ($.inArray(nearest.node.name, ['The French Froggies Project!','Xenopus as a chassis','Hormonal Communication','Modeling','GoldenBricks', 'Human Practice', 'The Team']) >=0 ){<br />
if (nearest.node.name!=_section){<br />
_section = nearest.node.name<br />
that.switchSection(_section)<br />
}<br />
dom.removeClass('linkable')<br />
window.status = ''<br />
}<br />
<br />
return false<br />
},<br />
clicked:function(e){<br />
var pos = $(canvas).offset();<br />
_mouseP = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
nearest = dragged = sys.nearest(_mouseP);<br />
<br />
if (nearest && selected && nearest.node===selected.node){<br />
var link = selected.node.data.link<br />
if (link.match(/^#/)){<br />
$(that).trigger({type:"navigate", path:link.substr(1)})<br />
}else{<br />
window.location = link<br />
}<br />
return false<br />
}<br />
<br />
<br />
if (dragged && dragged.node !== null) dragged.node.fixed = true<br />
<br />
$(canvas).unbind('mousemove', handler.moved);<br />
$(canvas).bind('mousemove', handler.dragged)<br />
$(window).bind('mouseup', handler.dropped)<br />
<br />
return false<br />
},<br />
dragged:function(e){<br />
var old_nearest = nearest && nearest.node._id<br />
var pos = $(canvas).offset();<br />
var s = arbor.Point(e.pageX-pos.left, e.pageY-pos.top)<br />
<br />
if (!nearest) return<br />
if (dragged !== null && dragged.node !== null){<br />
var p = sys.fromScreen(s)<br />
dragged.node.p = p<br />
}<br />
<br />
return false<br />
},<br />
<br />
dropped:function(e){<br />
if (dragged===null || dragged.node===undefined) return<br />
if (dragged.node !== null) dragged.node.fixed = false<br />
dragged.node.tempMass = 1000<br />
dragged = null;<br />
// selected = null<br />
$(canvas).unbind('mousemove', handler.dragged)<br />
$(window).unbind('mouseup', handler.dropped)<br />
$(canvas).bind('mousemove', handler.moved);<br />
_mouseP = null<br />
return false<br />
}<br />
<br />
<br />
}<br />
<br />
$(canvas).mousedown(handler.clicked);<br />
$(canvas).mousemove(handler.moved);<br />
<br />
}<br />
}<br />
<br />
return that<br />
}<br />
<br />
<br />
var Nav = function(elt){<br />
var dom = $(elt)<br />
<br />
var _path = null<br />
<br />
var that = {<br />
init:function(){<br />
$(window).bind('popstate',that.navigate)<br />
dom.find('> a').click(that.back)<br />
$('.more').one('click',that.more)<br />
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})(this.jQuery)</div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:54:43Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<h3 id="24hours">24h after injection</h3><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br />
<br />
<h3>48h after injection</h3><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br />
<br />
<br />
<h3>3 days after injection</h3<br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br />
<h3>4 days after injection</h3><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.</p><br />
<br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br />
<br />
<h2 id="GFPaid"><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br />
<br />
<h2 id="mCFP"><li>pCS2+ mCFP</li> </h2><br />
<br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br />
<br />
<h2 id="sfGFP"><li>pCS2+ sfGFP</li></h2><br />
<br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:53:36Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<h3 id="24hours">24h after injection</h3><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br />
<br />
<h3>48h after injection</h3><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br />
<br />
<br />
<h3>3 days after injection</h3<br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br />
<h3>4 days after injection</h3><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.</p><br />
<br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br />
<br />
<h2 id="GFPaid"><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br />
<br />
<h2 id="mCFP"><li>pCS2+ mCFP</li> </h2><br />
<br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br />
<br />
<h2 id="sfGFP"><li>pCS2+ sfGFP</li></h2><br />
<br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:51:01Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<h3 id="24hours">24h after injection</h3><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br />
<br />
<h3>48h after injection</h3><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br />
<br />
<br />
<h3>3 days after injection</h3<br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<h2 id="GFPaid"><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<h2 id="mCFP"><li>pCS2+ mCFP</li> </h2><br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<h2 id="sfGFP"><li>pCS2+ sfGFP</li></h2><br />
<br/><br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:49:02Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<br/><br />
<h3 id="24hours">24h after injection</h3><br />
<br/><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br/><br />
<br />
<h3>48h after injection</h3><br />
<br/><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/><br />
<br />
<br />
<h3>3 days after injection</h3><br />
<br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<br/><br/><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br/><br/><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<h2 id="GFPaid"><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<h2 id="mCFP"><li>pCS2+ mCFP</li> </h2><br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<h2 id="sfGFP"><li>pCS2+ sfGFP</li></h2><br />
<br/><br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:45:30Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
<br/><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<br/><br />
<h3 id="24hours">24h after injection</h3><br />
<br/><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br/><br />
<br />
<h3>48h after injection</h3><br />
<br/><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/><br />
<br />
<br />
<h3>3 days after injection</h3><br />
<br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<br/><br/><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br/><br/><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<h2 id="GFPaid"><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<h2 id="mCFP"><li>pCS2+ mCFP</li> </h2><br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<h2 id="sfGFP"><li>pCS2+ sfGFP</li></h2><br />
<br/><br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:44:07Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng×µl<sup>-1</sup> - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
<br/><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<br/><br />
<h3 id="24hours">24h after injection</h3><br />
<br/><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br/><br />
<br />
<h3>48h after injection</h3><br />
<br/><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/><br />
<br />
<br />
<h3>3 days after injection</h3><br />
<br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<br/><br/><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br/><br/><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<li><h2 id="GFPaid">pCS2+ mCitrine</h2></li><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<li><h2 id="mCFP">pCS2+ mCFP</h2></li> <br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<li><h2 id="sfGFP">pCS2+ sfGFP</h2></li><br />
<br/><br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:22:09Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple<a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li><a href="#mCit" style="text-decoration:none;">pCS2+ mCitrine</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li><a href="#mCFP" style="text-decoration:none;">pCS2+ mCFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li><a href="#sfGFP" style="text-decoration:none;">pCS2+ sfGFP</a>: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<h2 id="ElaGFP"><li>pCS2+ Elastase/sfGFP</li></h2><br />
<br/><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<h2 id="GFPaid"><li>pCS2+ GFP-aid</li></h2><br />
<br/><br />
<h3 id="24hours">24h after injection</h3><br />
<br/><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br/><br />
<br />
<h3>48h after injection</h3><br />
<br/><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/><br />
<br />
<br />
<h3>3 days after injection</h3><br />
<br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<br/><br/><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br/><br/><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<li><h2 id="GFPaid">pCS2+ mCitrine</h2></li><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<li><h2 id="mCFP">pCS2+ mCFP</h2></li> <br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<li><h2 id="sfGFP">pCS2+ sfGFP</h2></li><br />
<br/><br/><br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/Tadpole_injection1Team:Evry/Tadpole injection12012-09-26T18:10:49Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<center><h1>Characterization of plasmids in <i>Xenopus tropicalis</i></h1><br/></center><br />
<br/><br />
<p><b>In order to test ours plasmids we injected them into fertilized eggs, then we followed the fluorescent expression throughout time and space depending on promoter (ubiquitous or tissue specific).</b></p><br />
<br/><br/><br />
<br />
<p>A very simple<a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains with diagrams how to do the injections and how to take care of embryos and tadpoles. The experiment last 5 days, from an unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 48-50. The GFP (or any other fluorescent protein) is expressed few hours after the fertilization to the end of the week (<a href="#24hours" style="text-decoration:none;">see below</a>).</p><br />
<br/><br />
<p>Embryos and tadpoles grew up with their own vitellus, without the need to be fed during the week of experiment. Pictures were taken with the Zeiss stereomicroscope: SteREO Lumar V12 with the camera AxioCamMRm. The same protocol was used to inject eggs, and we injected 2.3 nl of plasmid at 100ng.µl-1 - embryos were stored at 21°C during the experiment week.</p><br />
<br/><br />
<p>The iGEM-Evry tem say a great thanks to Dr. Nicolas Pollet, Dr. Aurore Thelie and Lena Vouillot (PhD student) who taught us how to inject embryos, take care of tadpoles and how to use their microscope. They are from the <a href="http://www.issb.genopole.fr/">Institute of Systems & Synthetic Biology</a> of Evry in the <a href="http://indigene.issb.genopole.fr/">Metamophosys</a> group.</p><br />
<br/><br/><br />
<br />
<h2>Plasmids injected:</h2><br />
<br/><br/><br />
<br />
<ol><br />
<li><a href="#ElaGFP" style="text-decoration:none;">pCS2+ Elastase/sfGFP</a>: This plasmid contains the tissue specific promoter elastase and the fluorescent protein sfGFP, elastase is a promoter specific to pancreas. This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a> (ready to use in Xenopus). This plasmid was built from the eukaryotic plasmid (with elastase promoter) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a> and the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.</li><br />
Number of Plasmids injected: ~ 5.27E+7 <br/><br/> <br />
<br />
<li><a href="#GFPaid" style="text-decoration:none;">pCS2+ GFP-aid</a>: This plasmid contains the constitutive promoter CMV and the aid sequence of the aid system fused to GFP (Green Fluorescent Protein)(Nishimura et al., 2009). This Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812110">BBa_K812110</a> (ready to use in Xenopus). This plasmid was built from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick GFP-aid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>.</li><br />
Number of Plasmids injected: ~ 3.78E+7 <br/><br/> <br />
<br />
<li> pCS2+ mCitrine: This plasmid contains the constitutive promoter CMV and the fluorescent protein citrin (yellow). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">BBa_K812130</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCitrine (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">BBa_K812030</a>.</li><br />
Number of Plasmids injected: ~ 4.38E+7 <br/><br/> <br />
<br />
<li> pCS2+ mCFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein mCFP (Cyan Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">BBa_K812132</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick mCFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">BBa_K812032</a>.</li><br />
Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
<br />
<li> pCS2+ sfGFP: This plasmid contains the constitutive promoter CMV and the fluorescent protein sfGFP (super folded Green Fluorescent Protein). The plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">BBa_K812133</a> (ready to use in Xenopus) is from our pCS2+ eukaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a> with the BioBrick sfGFP (with the Kozak sequence) <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>.<br />
</li>Number of Plasmids injected: ~ 4.37E+7 <br/><br/> <br />
</ol><br />
<br />
<ol><br />
<br />
<li><h2 id="ElaGFP">pCS2+ Elastase/sfGFP</h2></li><br />
<br/><br />
Tadpoles are at stage 47. Their size is near 5 mm.<br />
<br/> <br />
<img src="/wiki/images/5/52/PCS2%2B_elastase_sfGFP.png" alt="perdu" width="880px"/><br />
<br/><br />
<p>We can see a sfGFP expression in a region close to the intestine and the biliar vesicle, and it is not auto-fluorescence. We suspect it to be the pancreas but as it is small in the tadpole it is not easy to testify. This injection will be done again in order to confirm our results.</p><br />
<br />
<li><h2 id="GFPaid">pCS2+ GFP-aid</h2></li><br />
<br/><br />
<h3 id="24hours">24h after injection</h3><br />
<br/><br />
Embryos are around <a href="https://2012.igem.org/Team:Evry/Stages">stage 20</a>. The neural fold is visible and the size of neurulas is near 1 mm <br/><br />
<img src="/wiki/images/c/c7/409GFP-aid%2Bcontrol.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<img src="/wiki/images/2/2e/409gfpaid2.jpg" alt="perdu" width="880px"/><br />
<br/><br />
<center><img src="/wiki/images/d/d6/409zstackGFP.gif" alt="perdu"width="400px"/><br/><br />
<i>z-stack of the embryo</i></center><br />
<br/><br/><br />
Some eggs expressed GFP-aid and some others did not meaning that the injection did not work for them or the GFP-aid was not express<br />
<br/><br/><br />
<br />
<h3>48h after injection</h3><br />
<br/><br />
Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 34-38</a> and move by intermittence, the size of tadpoles is near 2.5 mm<br/><br/><br />
<br />
<img src="/wiki/images/f/f8/509_gfpaid_1et2.JPG" alt="perdu" width="880px" /><br/><br />
<br />
<img src="/wiki/images/c/c8/509_gfpaid_ctrl.JPG" alt="perdu" width="880px" /><br/><br/><br />
<br />
<p>Despite a constitutive promoter in the plasmid (CMV), the expression of GFP-aid is localized in different tissues for each tadpole. We can hipothesize that the plasmids do not diffuse in the eggs because of the vitellus viscosity. This question was raised in one of the <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">modelling part.</a> <br/><br/><br />
<br />
<br />
<h3>3 days after injection</h3><br />
<br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage 41-42</a>. They swim and their size is near 4 mm.</p><br />
<br/><br/><br />
<p>From this tadpole stage an anaesthetic is required to take pictures of tadpoles, otherwise the light teases tadpoles, and it is impossible to take a picture.</p><br />
<br/><br/><br />
<br />
<img src="/wiki/images/d/d0/GFPAID%2BpNHK60Zstack-1.gif" alt="perdu" width="400px" /> <br />
<img src="/wiki/images/5/54/GFPAID-Zstack1-1.gif" alt="perdu" width="400px" /><br/><br/><br />
<br />
<p>z-stack pictures: pCS2+ CMV_GFP-aid, GFP expression is not localized in same tissue between tadpoles. For instance in the tadpole on the left picture, bones in the tail produced GFP; on the right picture the GFP expression is localized in the skin. In this animation the only part of the tadpole moving is the heart beatting (between the head and the stomac).</p><br />
<br/><br/><br />
<img src="/wiki/images/0/03/6.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/8/81/6.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The expression of GFP-aid is localized in different tissues for each tadpole, like the day before. GFP is present in same tissue, it means that plasmids stay in same cells.</p><br />
<br/><br/><br />
<br />
<h3>4 days after injection</h3><br/><br />
<p>Embryos are at <a href="https://2012.igem.org/Team:Evry/Stages">stage</a> 45-46. They swim and their size is near 5 mm.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/7/71/7.09_GFP-aid.JPG" alt="perdu" width="880px" /><br />
<img src="/wiki/images/f/f2/7.09_control.JPG" alt="perdu" width="880px" /><br />
<br/><br/><br />
<p>The GFP is still present in specific tissue but the intensity of the signal is decreasing. Plasmids may be damaged by cells, and/or the quantity of plasmids may have decrease in each cells which involded the diminution of GFP in each cells, after that it is more difficult to see GFP. Picture with the LSM 510 META Laser Scanning Microscope from Zeiss. A great thank to Dr Daniel Stockholm and <a href="http://www.genethon.fr/en/">Genethon</a> for using this microscope.<br />
</p><br />
<br/><br/><br />
<img src="/wiki/images/c/ce/Picture_genethon_GFP-aid_7.09.jpg" alt="perdu" width="880px" /><br/> <br />
<p>The tadpole 1 express GFP-aid in epidermic cells, whereas the tadpole 2 express GFP-aid in optical nerve,nostril nerve, tail muscle cells and branchial basket.<br />
</p><br />
<br/><br/><br />
<br />
<h2><li>pCS2+ mCitrine</li></h2><br />
<br/><br />
<p>Tadpoles have 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), because our lab does not have YFP filter for the mCitrine fluorescent protein the LSM 510 META Laser Scanning Microscope from Zeiss was used.</p><br />
<br/><br/><br />
<img src="/wiki/images/b/b6/Picture_citrine_7.09.jpg" alt="perdu" width="880px" /><br/><br />
The expression of mCitrine is localized in tail's muscles.<br />
<br/><br/><br />
<br />
<h2><li>pCS2+ mCFP</li> </h2><br />
<br/><br/><br />
<p>Tadpole 1 is 2 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 36-38</a>), the expression of CFP is localized in intestine. Tadpole 2 is 4 days (<a href="https://2012.igem.org/Team:Evry/Stages">stage 45-46</a>), the expression of CFP is localized in Branchial basket and intestine.</p><br />
<br/><br />
<img src="/wiki/images/5/58/CFP_tadpole.jpg" alt="perdu" width="880px" /> <br/><br/><br/><br />
<br />
<h2><li>pCS2+ sfgFP</li> </h2><br/><br/><br />
<br />
<img src="/wiki/images/2/27/PCS2%2B_sfGFP_20%2621_09.png" alt="perdu" width="880px" /> <br/><br/><br />
<p>The expression of sfGFP is present only in one out of the two embryo/tadpole. sfGFP is present only in few tissue as others reporters. sfGFP is expressed in tail's muscles and branchial basket for this tadpole.<br />
</p><br />
<br/><br/><br />
<br />
</ol><br />
<br />
<h2>Conclusion</h2><br/><br />
<p><br />
This page shows that our constructions were expressed in embryos and then in tadpoles. BioBricks parts above are considered as characterized. Nevertheless we expected an uniform expression of reporter with the CMV promoter. Colors fluorescent proteins was expressed in different tissue, one tadpole expresses the colors fluorescent protein in one to four different tissues, and tissue are different between tadpoles. <br/><br />
Hypothesis: The plasmid does not diffuse in the egg and stay in the same area, it means that depending on the injection area the plasmid would be in a part of the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.<br/> <br />
We think that we showed the tissue specificity of the elastase promoter (pancreas), but more experiment are necessary to confirm it. Four different reporters were characterized: mCitrine, CFP, sfGFP and the fused protein GFP-aid.<br />
Moreover the expression of reporters decreases throughout time, because plasmids are damaged throughout times but also because plasmids are shared between more cells and the quantity of plasmids decreases for each cell.<br/><br/><br />
</p><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/our-projectTeam:Evry/our-project2012-09-25T21:10:03Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<br />
<br><br />
<br />
<h1 align="center">Welcome to the French Froggies Home Page!<h1><br />
<br />
<h2>Introduction</h2><br />
<br />
<p>Dear visitor, welcome to the French Froggies project Homepage. This year, the Evry iGEM team pushed farther away the frontiers of synthetic biology bringing the frog as a new chassis for the iGEM competition. We have developed and characterized <em>the first parts for <i>Xenopus</i></em>, providing new tools for <em>modeling the physiology</em> of the animal and constructed the <em>first synthetic hormonal system</em> in a multicellular organism. </p><br />
<br />
<p>Have a look to the overview page to see a summary of each part of our project and access to every details and results we obtained.</p><br />
<br />
<br><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 400px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<table style="background:transparent";><tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><h3 style="text-align:center; padding-left:20px;"></u><a href="https://2012.igem.org/Team:Evry/Project"><center>Project overview</center></a></u></h3></td><br />
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<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
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<br><br />
<br />
<h1 align="center">Welcome to the French Froggies Home Page!<h1><br />
<br />
<h2>Introduction</h2><br />
<br />
<p>Dear visitor, welcome to the French Froggies project Homepage. This year, the Evry iGEM team pushed farther away the frontiers of synthetic biology bringing the frog as a new chassis for the iGEM competition. We have developed and characterized <em>the first parts for <i>Xenopus</i></em>, providing new tools for <em>modeling the physiology</em> of the animal and constructed the <em>first synthetic hormonal system</em> in a multicellular organism. </p><br />
<br />
<p>Have a look to the overview page to see a summary of each part of our project and access to every details and results we obtained.</p><br />
<br />
<br><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 400px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<table style="background:transparent";><tr><td><img style="padding-top:10px;padding-left:10px;" src="https://static.igem.org/mediawiki/2012/0/0f/French_frog.png" width=80px></td><br />
<td><h3 style="text-align:center; padding-left:20px;"></u><a href="https://2012.igem.org/Team:Evry/Project">Project overview></a></u></h3></td><br />
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<hr />
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<html><br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<!-- <p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> --><br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and intertissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multicellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <a href="https://2012.igem.org/Team:Evry/AIDSystem">An orthogonal hormonal system.</a> </p><br/><br />
<br />
<br />
<h2>The simple molecular strategy to build eukaryotic plasmid ready to use: </h2><br />
<br/><br/><br/><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br />
<br/><br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in Xenopus</h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage 48-50</a>. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>We injected 2.3 nl of plasmids at 100ng.µl-1 - embryos were stored at 21°C during all the experiment. pCS2+ GFP-aid: contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
<br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>We showed that our constructions express our reporters with the CMV promoter. These parts are considered characterized. Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Spectral variants of fluorescent proteins could be expressed in different tissues. Within one tadpole the fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.</P><br />
<P>Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.</P><br />
<P>Moreover the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell.</p><br><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:53:34Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis ?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
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<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
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<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
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<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
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<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis ?</a><br><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:51:43Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: What does it mean to be a chassis ?</a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/HumanPracticeTeam:Evry/HumanPractice2012-09-25T20:51:21Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<html><br />
<center><h1> Human practice: What does it mean to be a chassis ? </h1></center><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/3/32/Book_frog.png" align="center"></center><br />
<br />
<br><br><br />
<h2> Overview </h2><br />
<br />
<ul><br />
<p><li> <a href="https://2012.igem.org/Team:Evry/HumanPractice/Introduction"> Report </a>: Are you a chassis? [PDF version coming soon!]<br></p><br />
<p>For our human practice we decided to develop a philosophical investigation concerning the introduction of <i> Xenopus tropicalis </i> as a new chassis for the iGEM contest.</p><br />
<p> Here comes the map of our investigation, feel free to go where you like! <br></p><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/d/d5/Hi_Xenope!_3.jpg" alt="image not found" width="900" /><br />
<br />
<p><li> <a href="https://2012.igem.org/Team:Evry/HumanPractice/others"> Laws, debates and surveys </a> <br></p><br />
An annex summering the legal aspects of animal experimentation, the debates in La Paillasse and the two surveys which helped us start our investigation.</p><br />
<br />
</ul><br />
<br />
<br><br><br />
<br />
<br />
<script type="text/javascript">writeFooter()</script><br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/ModelingTeam:Evry/Modeling2012-09-25T20:50:13Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><h1>Modeling a tadpole: a multi-level approach</h1></center><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2012/6/62/MathFrog.png" width=200px></center><br />
<br />
<h2>Modeling a system on a complete organism</h2><br />
<br><br />
<p>This year, our team decided to tackle a challenging project: creating de-novo a new hormonal system in a vertebrate organism. In the modeling part of our work, we were interested in modeling the entire genetic and transport system in the host organism in order to understand the system better as well as to give indications to guide the development of the system in the wet part of our work.</p><br />
<br />
<br><br />
<p>Modeling a system at the organism level is not an easy task at all. Various approaches and hypothesis have to be used depending on the scale you want to look at and the question you ask. In our work, we used a large combination of classical synthetic biology modeling techniques to look at the system from the organism to the molecular level, using analytical and modeling techniques involving biochemical models, diffusion and transport models using simultaneously and in conjunction Ordinary Differential Equations (ODEs), Partial Differential equations (PDEs) and Agent Based simulations (AB).<br />
<br><br />
<h2>Preparing the work of the future generations on iGEMers working on complex organism</h2><br />
<br><br />
<p>When writing down our work, we have made an especial effort for our models and hypothesis to be very understandable, in order help the work of the future generation of iGEMers working on tadpoles, and on multicellular organisms in general. You can access general informations on the models by clicking on the ODE, PDE and AB simulations on the image below, and all the instructions are provided for you to run our simulations either on your own computer or directly in your web browser (!) using the full power of the Java Applets created using the Netlogo program.</p><br />
<p>We hope our work will be useful for other to learn, enjoy and create new models for their projects in the future!</p><br />
<br />
<h2>Parameters estimation</h2><br />
<br />
<p>A great part of our modeling work has been to find or estimate the values of the parameters used in the model. For a better readability, we created a special pages regrouping them all: <a href="https://2012.igem.org/Team:Evry/parameters">here</a>.</p><br />
<br />
<h2>Overall map of models</h2><br />
<br><br />
<br />
<p>This schematic represents the different parts of the models we have created, as well as general information on the modeling methods used in these models. </p><br />
<h4>Clicks on the different elements of this image to access the different models:</h4><br />
<br />
<br ><br />
<br><br />
<br />
<center><br />
<br />
<script type="text/javascript" src="https://2012.igem.org/Team:Evry/wz_jsgraphics.js?action=raw"></script><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Evry/mapper.js?action=raw"></script> <br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/6f/Schematic_modelingV3.png" width="945" height="829" border="0" class="mapper" usemap="#map" align="center" /><br />
<br />
<map name="map"><br />
<area shape="rect" class="noborder icolor474747" coords="240,120,665,210" href="ODE_model" /><br />
<area shape="rect" class="noborder icolor474747" coords="240,225,665,315" href="auxin_pde" /><br />
<area shape="rect" class="noborder icolor474747" coords="240,330,665,450" href="Auxin_diffusion" /><br />
<area shape="rect" class="noborder icolor474747" coords="240,495,665,600" href="auxin_production" /><br />
<area shape="rect" class="noborder icolor474747" coords="240,615,665,690" href="auxin_degradation" /><br />
<area shape="rect" class="noborder icolor474747" coords="240,705,665,795" href="plasmid_splitting" /><br />
<area shape="rect" class="noborder icolor474747" coords="710,144,805,189" href="parameters" /><br />
<area shape="rect" class="noborder icolor474747" coords="710,365,805,410" href="parameters" /><br />
<area shape="rect" class="noborder icolor474747" coords="710,525,805,570" href="parameters" /><br />
<area shape="rect" class="noborder icolor474747" coords="710,630,805,675" href="parameters" /><br />
<area shape="rect" class="noborder icolor474747" coords="710,735,805,780" href="parameters" /><br />
<area shape="rect" class="noborder icolor474747" coords="810,430,955,480" href="model_integration" /><br />
<area shape="rect" class="noborder icolor474747" coords="11,720,131,795" nohref="no_ref" /><br />
</map><br />
</center><br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i> An introduction to agent-based modeling: Modeling natural, social and engineered complex systems with NetLogo</i>, Wilensky, U., & Rand, W. (in press), Cambridge, MA: MIT Press</li><br />
</ol><br />
</div><br />
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<center><h1>GoldenBrick: A new biobrick cloning format</h1></center><br />
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<h3 style="text-align: center; padding-top:10px;"></u>Warning:</u></h3><br />
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<p align=center>These results are very preliminaries, but if the results match our expectancies, we are going to publish the technique soon. Please don't use any of the results and sequences published in this page without our permission.</p><br />
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<h3 style="text-align: center; padding-top:10px;"></u>Call for collaboration:</u></h3><br />
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<p align=center>Do you like this idea? Would you like to contribute with us to demonstrate the interest of the technique for iGEM? Contact us at igemevry2012@gmail.com and we will discuss how you can contribute to this big project!</p><br />
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<h1>Introduction</h1><br />
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<h2>Context</h2><br />
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<br />
<div class="thumb tright"><div class="thumbinner" style="width:302px;"><a href="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" width="300" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 1: Picture of one of our genetically modified tadpole (z-stack)</div></div></div><br />
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<p>Cloning is probably the most tedious and less rewarding task when engineering living entities through synthetic biology. With the development of the restriction enzymes and efficient DNA ligases, assembling small and large DNA pieces has become a common practice in biology, but as everyone is using their own sets of enzyme and assembly method, making large DNA constructs is still a challenge today. The first indisputable advance in the domain came with the invention of the first standardized DNA cloning method: the BioBricks []. The standard BioBrick format (BBF RFC 10) make possible to clone together all kind of genetic parts (called "bricks") using only four different enzymes: EcoRI, XbaI, SpeI and PstI. But the real power of BioBrick standardization lays in the possibility to build database made of these DNA pieces that anyone can be assembled through a standard method. The PartsRegistry today contains several thousands of described and characterized biological parts [] and is a sources of inspiration for thousands of biologists around the world.<p><br />
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<p>If biobricks have opened new perspectives for engineering organisms and have proven its efficiency over a decade, this technique is limited by the fact that the fragments can only be assembled one by one. On the top of this, the process is very difficult to automatize, because of the number of DNA purification step required. IGEMers today spend most of their time assembling genetic parts together, leaving little time for characterizing them and testing their systems.</p><br />
<br />
<p>Several cloning techniques capable of assembling multiple fragments at the time have been invented since the rise of the Biobrick format. The most popular one is probably the one known as the Gibson method [] that can be used to assemble up to four fragments at the time on a regular basis. The Golden Gate technique [] (>20 fragments at the time) and its new evolution, the MoClo [] (47 fragments in two times reported) are leading the way of another kind of cloning technique based on type II restriction enzymes. More efficient than the Gibson cloning, the Golden Gate also have the huge avantage that it can assemble more parts at the time, no matter their size. Moreover, Golden Gate do not imply any DNA modifications, which noticeably reduce the probability of having a mutation at the ligation scar.</p><br />
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<p>If these new techniques dramatically speed-up the assembly of DNA pieces, to our knowledge, no reported work has been carried on standardizing these methodologies. So far, biologists using Golden Gate engineer new overhangs and create a new library each time they are making a different construct.</p><br />
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<h2>From Golden Gate and BioBricks to GoldenBricks</h2><br />
<br />
<p>The BioBricks are a collection of parts that can be assembled one by one in a generic way. Golden Gate is a technique that enable the assembly of several tens of parts simultaneously. Taking inpiration from both technique, the 2012 Evry iGEM Team invented and developped a new methodology called GoldenBricks, merging the power of the two techniques while keeping the compatibility toward the old RFC 10 BioBrick format and preserving the possiblilty to make standard GoldenGate assembly simultaneously with GoldenBrick assembly, using the same vectors.<p><br />
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<p>GoldenBricks technique is a one-shot cassette construction using DNA parts coming either from a plasmid distribution or from PCR product, engaging 5, 7 and possibly more different parts. Moreover, GoldenBricks works for both eukaryotes and procaryotes DNA construction with the very same protocol. If a non classical assembly is required (as for testing the strength of a terminator) a new "split construction" method based on standard plasmids make possible to assemble parts in a non classical way.</p><br />
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<h2>Perspectives</h2><br />
<br />
<p>Such technique offers great perspectives for the future of iGEM and of the PartsRegistry in general. First, it would make possible the fast and simple assembly of complete cassettes using less expensive equipment, less toxic chemical and less sequencing runs than the BioBrick assembly. Second, it would make fast and efficient cloning accessible for both researchers as well as for less experimented users, such as high school iGEMers or biohackers thanks to the simplicity of its protocol. Third, the creation of large expression cassettes using this method is cheaper and faster than synthesizing the construct at the present day, which would guaranty the interest for a DNA database compared to <i>de-novo</i> synthesis for the years to come. And last, this method is a lot more easy to automatize unlike the RFC 10 biobrick format.</p><br />
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<h1>Theory</h1><br />
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<h2>Requirements for the development of the new standard</h2><br />
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<p>In the continuation of what we said previously and in order to stay in the progression of the methodology we have tried in this work to stay as close as possible to biobrick format, to keep GoldenBrick parts compatible with the RFC 10 standard assembly, while opening new perspectives to increase the speed and the easiness of cloning. These constraints imposed several requirements for this new standard.</p><br />
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<p>First, the new assembly method should:</p><br />
<ol><br />
<li>Keep the compatibility with the current BBF RFC 10 standard</li><br />
<li>Be compatible with a database approach</li><br />
<li>Minimize the number of different prefixes and suffixes</li><br />
<li>Use the standard registry plasmids and negative cloning control</li><br />
</ol><br />
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<p>To improve the cloning speed, we would also like to:</p><br />
<ol start=5><br />
<li>Make one step Golden Gate cassette cloning possible</li><br />
<li>Allow to check the construct with a single enzyme digestion</li><br />
<li>Improve the compatibility with Gibson by removing the NotI site</li><br />
<li>Provide a solution for non standard assemblies</li><br />
</ol><br />
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<p>As we will demonstrate, the GoldenBrick format we have established meet all these requirement. To understand on what basis we have created it, we are now going to explain in this section the principles that led the design of this format before we analyze the sequences of the GoldenBrick prefixes and suffixes and finally discuss of what can be done with this technique.<br />
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<h2>A brief introduction on type II restriction enzymes</h2><br />
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<p>Golden Gate cloning, (also known as type II restriction enzyme cloning) rely on a specific familly of enzyme (known as the type II enzymes) very different from the ones we are used to. The characteristic of type II enzymes from the cloning point ov view is that these enzymes cut the DNA outside their recognition locus, no matter the sequence of the place where they cut. A very small subtype of them can be used for cloning purpose, since they generate overhangs on one side of the recognition site only. The ones used for Golden Gate and MoClo cloning are BsaI and BbsI.</p><br />
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:202px;"><a href="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" width="200" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 2: The recognition pattern of BsaI and cutting site.</div></div></div></div><br />
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<p>The fact that the enzyme cuts outside its recognition site gives them two significant advantages:</p><br />
<ul><br />
<li>You can generate as many different overhang you want with a single enzyme within the same tube</li><br />
<li>You can engineer your fragment so that it cannot be cutted again after it has ligated.</li><br />
</ul><br />
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<p>Thanks to this last point, it is possible to cut and ligate the DNA in the mean time within the same tube (BsaI works in the T4 ligase buffer), and do not require any DNA purification. For this reason, the ligation is acheived with a better yeild and with less mutation than with traditional type I enzymes cloning methods.</p><br />
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Figure 3: Principle of the Golden Gate cloning method.</div></div></div></div><br />
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<p>Since the small cut BsaI fragment can re-ligate with its mother strand, in order to improve the yield, the assembly is a lot more efficient when using a cycling between digestion and ligation phases in a thermocycler alternating between 37°C and 16°C, which drives the equilibrium towards the formation of the product.</p><br />
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<h2>Analysis of a classical synthetic biology constructions</h2><br />
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<p>A traditional construct in synthetic biology is made of the repetition of the following elements:</p> <br />
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:402px;"><a href="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" width="400" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 4: Schematic of the different elements assembly in a standard synthetic biology device.</div></div></div></div><br />
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<p>At the moment, GoldenBrick only allow the assembly of a single cassette at the time. In order to create the full system, the assembly of the different cassettes together have to be conducted afterwards with the biobrick assembly, Gibson assembly - that is very efficient to assemble long fragments - or as we will discuss, ligation independant cloning []. Focusing closer on a single cassette, we can notice that we have 4 different kinds of junctions:</p><br />
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<br /><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 5: In a single cassette, we can notice 4 different kinds of junctions. If we want the repetition of the {RBD-CDS} unit to be possible, the prefix of the RBS have to be compatible with the suffix of the gene.</div></div></div></div><br />
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<p>As we can notice on this picture, there is 4 different kinds of junctions in a casette of device. In Golden Gate, the junctions have the form of a DNA overhang that we can freely engineer thanks to the capacity of Type II enzymes to cut outside its regognition site, no matter what sequence is there. Therefore, we should engineer 4 different overhangs. The first overhang is dedicated to the ligation of the plasmid suffix with the promoter prefix (OH1). Similarely, the 5th one is dedicated to ligate the terminator suffix with the backbone prefix (OH5). In a bacterial device, the scar between the RBS and the gene (OH3) has to be very well controlled because the distance between the two elements is critical for correct protein expression. This scar will remain the same than in the biobrick assembly, but it will be generated by a type II enzyme as for the others overhangs. Finally, the overhang that link the RBS prefix to the promoter suffix (OH2) and the protein suffix to the terminator prefix (OH4) have to be compatible to make possible the repetition of several {RBS-Protein} units in a single cassette (OH2=OH4). We will see later how to control this repetition.</p><br />
<br />
<p>To conclude, the necessity for 4 different overhangs implies to create 5 different prefixes and 5 different suffixes depending on the function of the DNA elements.</p><br />
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<p>In addition, we would like the scar generated by the assembly of all the different elements to be digestible by a single enzyme. This would enable to control whether all the elements we intended to insert in the construct has been assembled before sequencing the casette. As we will see later this element will be critical for the selection of the clone that have inserted all the genes we want. This is why the overhangs 1, 2, 4 and 5 has been engineered in the middle of a BbsI site. When two pieces ligates, they recreate a BbsI restriction site, and we can check the correct insertion of a given gene in the construct by simply digesting with BbsI and checking for the size on a gel.</p><br />
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<p>One last condition to fulfil to get a RFC 10 compatible brick after GoldenBrick cloning is to leave no illegal restriction site inside the brick after the assembly of the cassette. However, we should keep the as similar as possible the biobrick extensions around each elements leaving the 4 usuals enzyme restriction sites, so that the brick can be still assembled using biobrick method. The only drawback we didn't managed to overcome is that if several GoldenBrick parts are assembled with RFC 10 standard, they cannot be assembled together using GoldenGate afterwards, because the biobrick assembly will leave illegal BsaI site inside the casette.</p><br />
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<h2>The proposed new set of Golden Bricks extension</h2><br />
<br />
<p>Having all these design principles in mind and meeting the requirements we have proposed, we designed the new GoldenBrick prefix and suffix with the following sequences:</p><br />
<br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:635px;"><a href="/File:GoldenBricks.png" class="image"><img alt="" src="/wiki/images/e/ed/GoldenBricks.png" width="633" height="286" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GoldenBricks.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Figure 6: Sequence and design of the different prefixes and suffixes used in the GoldenGate cloning method. The presence of the EcoRI, XbaI, SpeI and PstI keep the compatibility with Biobrick cloning while the BsaI site enable single shot GoldenGate assembly. The BbsI sites serve as a single digestion control.</div></div></div></div><br />
<br />
<p>This design fulfil all the requirements enumerated before and ensure that the different elements will be assembled in the correct order. We also generate a scar that can be digested afterwards with the Bbsi enzyme, so that the correct insertion of the different elements can be check afterwards by doing a single digestion. This also enable the polymerization of the {RBS-CDS} units, as we will discuss in the next paragraph.</p><br />
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<h2>Control over the polymerization</h2><br />
<br />
<p>The only difference with a standard GoldenBrick protocol is the capacity for the RBS-protein cassette to polymerize. This polymerization capacity makes the power of the technique because it enable to make polysistronic mRNAs no matter what gene is inserted. However, a control have to be achieved oved it in order to control when to create polysistrons and when we want a single gene to be inserted.</p><br />
<br />
<p>In order to acheive such a control, several parameters in the protocol will move the balance from mono-systrons to poly-sistrons. The first and the most obvious one is the stoechimoetry of the different elements. If the promoter and terminators are in large excess compared to the RBSs and the gene, the balance will be in favour of mono-sistrons. On the contrary, if the amount or RBSs and gene in dominant, the cassette will tends to contain two or more genes inserted inside.</p><br />
<br />
<p>On the other hand, the number of cycle and the ligation time will also influence the polymerization. Poly-sistrons are not likely to appear when the number of cycle is small and the ligation time short, but they will become more and more important as we increase these parameters.</p><br />
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<p>Most of the work on GoldenBrick by our team is now focused in optimizing the protocol. We will release soon a set of standard protocols for mono and polysistrons optimized to reduce the polydispersity of the polymerization lenght.</p><br />
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="/File:GoldenBrick-polymerization.png" class="image"><img alt="" src="/wiki/images/1/10/GoldenBrick-polymerization.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GoldenBrick-polymerization.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 8: a. The vector, promoter, and terminator are in excess: the assembly produces mostly monosistrons. b. The protein sequences and RBS are in excess: the assembly produces mostly polysistrons.</div></div></div></div><br />
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<h2>Assembly procedure</h2><br />
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<div class="thumb tright"><div class="thumbinner" style="width:302px;"><a href="/File:GOldenbrick_global_thumb.png" class="image"><img alt="" src="/wiki/images/thumb/f/f3/GOldenbrick_global_thumb.png/300px-GOldenbrick_global_thumb.png" width="300" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GOldenbrick_global_thumb.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 9: Summary of the GoldenBrick procedure</div></div></div><br />
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<p>Assembling parts using the GoldenBrick method is a lot easier than for BioBricks and also 5 to 10 times faster. We are now going to explain the procedure from the theoretical point of view. For the complete protocol, you should refers to the material and method section.</p><br />
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<p>Because it requires a very small amount of DNA, the plasmid source can be taken directely from a DNA distribution suck as the partsregistry distribution, without requiring the need of a DNA preparation before. Once the plasmid are resuspended, the experimentalist mix them together in a mix containing the BsaI enzyme and T4 ligase and put them in the thermocycler for 5 hours. In the end, the product can be directely transformed and plated. A first round of screening sor the good polymerization length can be made using colony PCR with the standard VF2 and VR primers. If required, a second step of screening can be made after minipreping using BbsI. Given the very small rate of mutation, only two clones with the good digestion or colony PCR signature can be sent for sequencing; they will contain the correct construct with a very high probability.</p><br />
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<p>This technique however requires to screen an important number of clones because we don't have a total control of what is assembling inside the cassette, because of the freedom we left for the RBS-Protein entities to polymerize. We will show in the next paragraph how we can gain a relative control over it. To reduce the costs of screening, we encourage the use of colony PCR oven mini-preping and digestion when possible.</p><br />
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<h1>Other advantage of GoldenBricks over any other standard cloning techniques</h1><br />
<br />
<h2>Non standard assembly and "split-construct" vectors</h2><br />
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<p>If you are willing ot create a non standard assembly or to control very precisely RBS-protein pairing over a polysistrons, you might like to create the construct in two times and assemble them together in a second step. This is why we have created the "split-construct" vectors. The split-contruct vectors are like the goldenbrick vectors except that they contain the OH2-OH5 or the OH1-OH4 overhangs instead of the OH1-OH5 overhangs as the GoldenBrick plasmid.</p><br />
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<p>These vectors enable the assembly of shorter casette containing only a promoter, and a serie of RBS-protein for the "split-construct" 1 vector, or RBS-protein terminator for the "split-construct" 2 vector. This way, if a non standard biobrick assembly have to be carried in between the two pieces or if a long polysistron have to be created, they can be assembled using the standard EcoRI, XbaI, SpeI, PstI cloning, but several cloning step would have been saved by using GoldenBricks compared to a standard assembly.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 10: Description of the construction of a non classical cassette for creating a device that test the strength of a terminator using split-vector GoldenBricked mixed with classical or Gibson assembly. The GFP and RFP rations are measure to evaluate the strengh of a terminator the classical way. Two cloning steps are avoided using this procedure.</div></div></div></div><br />
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<h2>GoldenBrick make standard part DNA shuffling possible</h2><br />
<br />
<p>Aside from the dramatic shortening of the cloning time, the decrease of the costs and the easiness of GoldenBricks over Biobricks, one of the most noticeable improvement is undoubtedly the possibility to make DNA shuffling using a standard format. Lets take the example of a complex system such as a toogle-switch, in which you would like to optimize the expression of the protein repression of its two component to place the system in its working domain.</p><br />
<br />
<p>Using the Biobrick format, you will probably have to make tens of different constructs with different RBS to kind a condition in which the toogle-switch will work. Using GoldenBricks, you can make all of them in a single tube, in a single step. Moreover, you may eventually find a way to screen them directely after the cloning avoiding the need of purifying and sequence all the clones that couldn't work. The figure 11 shows in theory how one can proceed to such experiment design.</p><br />
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 11: Description of a DNA shuffling experiment in order to optimize the expression of a single gene. 4 Promoters and RBSs are mixed together in a single tube and give rise to 16 construct in a single tube in a single step. The clone expression the enzyme at the correct level can be screened directly on the transformation plate.</div></div></div></div><br />
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<p>Many other use of DNA shuffling can be find in the literature especially for gene expression optimization and screening for working enzymes. We expect that DNA shuffling over standard parts is going to find many applications in the future of iGEM projects.</p><br />
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<h2>GoldenBrick is still compatible with Biobrick format</h2><br />
<br />
<p>If the situation requires the part to be assembled with the standard RFC 10 format, the new GoldenBrick parts can be assembled the very same way than BioBricks. The only limitation is that BioBrick assembly of GoldenBrick parts leave a scar containing the BsaI restriction site inside, and the produced parts cannot be reassembled with other parts using Golden Gate. No simple way of fixing this issue has been found.</p><br />
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<h2>GoldenBricked plasmids are compatible with standard Golden Gate assembly</h2><br />
<br />
<p>The GoldenBricked plasmids can be used to assemble contructions using classical Golden Gate. This option has been tested in our team and it works fine. (result not presented here)</p> <br />
<br />
<h2>GoldenBrick also assemble PCR products and shorten the assembly time</h2><br />
<br />
<p>Someone than want to create a construct not having the time or the need of cloning his gene into a vector first can stil assemble them using GoldenBrick. Although this pracice wouldn't be encouraged for standards parts, it can become really usefull when trying libraries of different genes in order to find a working one. GoldenBricked parts in plasmid and PCR GoldenBricked parts can be mixed together and assembled the very same way. The user will just have to take care to submit his working parts afterwards. This is why we recommend to amplify new parts flanked by its GoldenBricks prefix and suffix, mix them with the GoldenGate assembly mix in one hand, and digest them in EcoRI and PstI on the other in order to ligate them in pSB1C3 forming the GoldenBrick.<br />
<br />
<h2>Classical GoldenGate can be mixed GoldenBrick assembly to create fusion proteins</h2><br />
<br />
<p>GoldenGate in general and GoldenBricks in particular are very efficient to assemble fusion proteins, because one can design every overhang he wants and therefore fuse proteins without making any scar. New fusion proteins can be created while beeing assembled in the standard GoldenBrick technique with custom made scar designed by the experimentalist. A new fusion protein made of several domains can be assembled in the middle of a polysistron without having to make the fusion protein first before cloning.</p><br />
<br />
<h2>Finally, GoldenBricks works with eukaryotes chassis as well as with prokaryotes</h2><br />
<br />
<p>As we have seen all along this article, this design of GoldenBricks have been design for procaryotes, but it can work very well for eukaryotes. In eukaryotes, promoters can include or not a 5'UTR in their sequence to enhance the protein expression. In the case there is no 5'UTR, we can use the same prefix and suffix than the prokaryote promoters and include the 5'UTR with the prokaryotes prefix and suffix.</p><br />
<br />
<p>In order to make polysistrons, we can use an Intermediate Ribosome Entry Site (IRES), cloned between an prokaryote RBS prefix and suffix.</p><br />
<br />
<p>Finally, the polyA and the polyA capping cassette with a polymerase terminator can be flanked by the prokaryotes terminators sites. In the case one don't want to include a polyA capping, he can use the "split construct" 1 or in the case there is not terminator, the one on the plasmid will take care of stopping the RNA polymerase extension.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 12: GoldenBricks sites for eukaryotes constructions.</div></div></div></div><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of the library</h2><br />
<br />
<p>In order to test the GoldenBrick technique efficiently, and for easiness of screening and making statistics, we have developed a set of GoldenBricked parts (see Material & Methods) with fluorescent proteins as a screening set. The parts have that we have created successfully are the following:<br />
<br />
<ul><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812050">K812050:</a> A GoldenBricked version of pSB1C3 with J04450 as negative cloning control (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812051">K812051:</a> A GoldenBricked version of pSB1K3 with J04450 as negative cloning control (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812053">K812053:</a> A GoldenBricked version of the strong RBS B0034 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812054">K812054:</a> A GoldenBricked version of the RFP E1010 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812055">K812055:</a> A GoldenBricked version of the terminator B0015 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812056">K812056:</a> A GoldenBricked version of the pLac R0010 promoter (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812057">K812057:</a> A GoldenBricked of an sfGFP protein (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812058:</a> A GoldenBricked of medium strenght RBS J61107 (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812059:</a> A GoldenBricked of week RBS J61117 (status=constructed)</li><br />
</ul><br />
<br />
<p>All the parts written here have been successfully constructed and sequenced and the ones marked as "sent" has been sent to the PartsRegistry.</p><br />
<br />
<h2>Creation of a simple cassette</h2><br />
<br />
<p>We have started experimenting on the GoldenBrick assembly method. For the moment, the results are not optimal so we are not publishing them yet. Betters results are to come since we are optimizing the protocol now.</p><br />
<br />
<h2>Working with regular GoldenGate using GoldenBricks vectors</h2> <br />
<br />
<p>In our way of setting up new protocols and in order to assemble the fusion proteins we needed for our auxin system, we carried out two assemblies of fusion proteins using Golden Gate in our Golden Gated vectors. This paragraph reports the construction of the IaaH-Pep2A-IaaM fusion protein in the GoldenBrick plasmid K812050 using standard Golden Gate protocol. This protocol hadn't been optimized at the moment. However, out of 16 cloned on which we did colony PCR, 10 of them had the good size and out of the 3 sequenced, they were all correct.</p><br />
<br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:702px;"><a href="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" width="700" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 13: Colony PCR of 15 clones from the transformation plate. The ones marked with a star has been sequenced and were corrects. The one with and exclamation mark was sequenced and had only one of the CDS inserted.</div></div></div></div><br />
<br />
<p>This experiment demonstrates the possibility of doing Golden Gate in the GoldenBricks plasmids K812050 (and similarly K812051).</p><br />
<br />
<br />
<h1>Material and methods</h1><br />
<br />
<h2>Construction of the library</h2><br />
<br />
<p>The GoldenBricked parts were constructed amplifying the corresponding biobricks using primers flanked by the appropriate GoldenBricks extensions, using Phusion (Thermo Scientific) or Q5 (NEB) DNA polymerase in a 30 cycle reaction. The PCR product was PCR purified and then digested using FastDigest® EcoRI and PstI (Thermo Scientific). The standard plasmid pSB1C3 (or pSB1K3) were digested similarely. Both vectors and inserts were PCR purified and then ligated using T4 DNA ligase (Thermo Scientific) before being transformed with home made competent cells (chemical or electroporation).<p><br />
<br />
<p>The either red or non red colonies were minipreped and then digested with BsaI for screening. All the clones that had the correct digestion pattern were sequenced and gave the correct sequence.</p><br />
<br />
<p>The RBSs were synthetized in the form of two mathing oligos. A mixture of 10 µM of the two oligo was incubated at 98°C for 2 min and went down to 4°C with a ramp of -1°C/s. They were then PCR purified, digested and PCR purified again before beeing ligated in the same way.</p><br />
<br />
<h2>Assembly protocol</h2><br />
<br />
<p>The protocol given here han't been optimized yet. We are only starting the development of this new technology. It is given as a matter of indication. Some optimized protocol will be available soon.</p><br />
<br />
<p>About 75 ng of each plasmid (more accurate ratios will be optimized) were mixed together with 2.5 units of BsaI enzyme (NEB) and 15 units of T4 DNA ligase (Thermo Scientific) in a total volume of 15 µL of standard T4 ligation buffer. The tubes were then placed in the thermocycler with with the following steps:</p><br />
<br />
<ol><br />
<li>37°C, 2 min (digestion)</li><br />
<li>16°C, 5 min (ligation)</li><br />
<li>Goto 1, 50 times</li><br />
<li><ul><li>Option 1: if the construct does not contain any BsaI site: 50°C 5 min (reduce the background with a final digestion)</li><br />
<li>Option 2: if the construct contains BsaI site: 16°C 2h (reconstitute the BsaI site)</li></ul></li><br />
<li>80°C, 5 min (heat inactivation)</li><br />
</ol><br />
<br />
<p>5 µL of the product was transformed with 50 µL of home made chemically competent cells and recovered for 1 hour. The cells were concentrated and plated.</p><br />
<br />
<h1>Perspectives for GoldenBricks and the PartsRegistry</h1><br />
<br />
<p>When the complete procedure will be established, our team will submit an BBF RFC request for the registry. If the RFC get accepted and if the tenant of the PartsRegistry thinks this is a valuable format, we can think of migrating the registry to this new format. This can be done in two ways: the first one is to ask every user to submit their new part in the new GoldeBrick format, and the second is to take advantage of the huge community working on the PartsRegistry and especially the iGEM comptetion. In order to migrate the parts faster, we can add in the requirements for the one of the medals for the teams to resubmit already exsiting parts in the new format. With an average of 200 teams in the years to come and asking each of them to resubmit 2 goldenbricked parts, we can cover the 2000 most useful parts within 2 or 3 years, because the teams will goldenbrick the parts they are interested in for their assembly, so they will probably always re-submit more than 2.</p><br />
<br />
<p>We beleive that implementing such a method in the partsregistry will speed up the assembly process and give a new shine to this database that severely suffers from the gene synthesis and PCR based methods competition. This will leave more time for researchers and iGEMers for characterizing the parts, and the partregistry will also benefits from an increase of parts characterization, which is very beneficial to the community. Overhall, the idea of standardized parts that tends to loose its strengh with the time will gain again in interest in the long run.</p> <br />
<br />
<h1>Conclusion</h1><br />
<br />
<p>Our team this year have created a new cloning format for the PartsRegistry that dramatically speed up the cloning time and open new perspective such as simple protein fusion methods and DNA shuffling that were not possible using the current BioBrick format. At the moment, we are working very hard to demonstrate its possibilities and come up with an efficient and reliable protocol. If the process is found to be efficient, we will submit an RFC request to the PartsRegistry and propose that GoldenBrick progressively substitute to the current Biobrick format, which will renew the interest for people on standardized biological parts, seriously challenged by gene synthesis and PCR based methods today.</p><br />
<br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>An introduction to agent-based modeling: Modeling natural, social and engineered complex systems with NetLogo</i>, Wilensky, U., & Rand, W. (in press), Cambridge, MA: MIT Press</li><br />
</ol><br />
</div><br />
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<h1>GoldenBrick: A new biobrick cloning format</h1><br />
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<p align=center>These results are very preliminaries, but if the results match our expectancies, we are going to publish the technique soon. Please don't use any of the results and sequences published in this page without our permission.</p><br />
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<h3 style="text-align: center; padding-top:10px;"></u>Call for collaboration:</u></h3><br />
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<p align=center>Do you like this idea? Would you like to contribute with us to demonstrate the interest of the technique for iGEM? Contact us at igemevry2012@gmail.com and we will discuss how you can contribute to this big project!</p><br />
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<br />
<h1>Introduction</h1><br />
<br />
<h2>Context</h2><br />
<br />
<br />
<div class="thumb tright"><div class="thumbinner" style="width:302px;"><a href="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" width="300" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/5/54/GFPAID-Zstack1-1.gif" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 1: Picture of one of our genetically modified tadpole (z-stack)</div></div></div><br />
<br />
<p>Cloning is probably the most tedious and less rewarding task when engineering living entities through synthetic biology. With the development of the restriction enzymes and efficient DNA ligases, assembling small and large DNA pieces has become a common practice in biology, but as everyone is using their own sets of enzyme and assembly method, making large DNA constructs is still a challenge today. The first indisputable advance in the domain came with the invention of the first standardized DNA cloning method: the BioBricks []. The standard BioBrick format (BBF RFC 10) make possible to clone together all kind of genetic parts (called "bricks") using only four different enzymes: EcoRI, XbaI, SpeI and PstI. But the real power of BioBrick standardization lays in the possibility to build database made of these DNA pieces that anyone can be assembled through a standard method. The PartsRegistry today contains several thousands of described and characterized biological parts [] and is a sources of inspiration for thousands of biologists around the world.<p><br />
<br />
<p>If biobricks have opened new perspectives for engineering organisms and have proven its efficiency over a decade, this technique is limited by the fact that the fragments can only be assembled one by one. On the top of this, the process is very difficult to automatize, because of the number of DNA purification step required. IGEMers today spend most of their time assembling genetic parts together, leaving little time for characterizing them and testing their systems.</p><br />
<br />
<p>Several cloning techniques capable of assembling multiple fragments at the time have been invented since the rise of the Biobrick format. The most popular one is probably the one known as the Gibson method [] that can be used to assemble up to four fragments at the time on a regular basis. The Golden Gate technique [] (>20 fragments at the time) and its new evolution, the MoClo [] (47 fragments in two times reported) are leading the way of another kind of cloning technique based on type II restriction enzymes. More efficient than the Gibson cloning, the Golden Gate also have the huge avantage that it can assemble more parts at the time, no matter their size. Moreover, Golden Gate do not imply any DNA modifications, which noticeably reduce the probability of having a mutation at the ligation scar.</p><br />
<br />
<p>If these new techniques dramatically speed-up the assembly of DNA pieces, to our knowledge, no reported work has been carried on standardizing these methodologies. So far, biologists using Golden Gate engineer new overhangs and create a new library each time they are making a different construct.</p><br />
<br />
<br />
<h2>From Golden Gate and BioBricks to GoldenBricks</h2><br />
<br />
<p>The BioBricks are a collection of parts that can be assembled one by one in a generic way. Golden Gate is a technique that enable the assembly of several tens of parts simultaneously. Taking inpiration from both technique, the 2012 Evry iGEM Team invented and developped a new methodology called GoldenBricks, merging the power of the two techniques while keeping the compatibility toward the old RFC 10 BioBrick format and preserving the possiblilty to make standard GoldenGate assembly simultaneously with GoldenBrick assembly, using the same vectors.<p><br />
<br />
<p>GoldenBricks technique is a one-shot cassette construction using DNA parts coming either from a plasmid distribution or from PCR product, engaging 5, 7 and possibly more different parts. Moreover, GoldenBricks works for both eukaryotes and procaryotes DNA construction with the very same protocol. If a non classical assembly is required (as for testing the strength of a terminator) a new "split construction" method based on standard plasmids make possible to assemble parts in a non classical way.</p><br />
<br />
<h2>Perspectives</h2><br />
<br />
<p>Such technique offers great perspectives for the future of iGEM and of the PartsRegistry in general. First, it would make possible the fast and simple assembly of complete cassettes using less expensive equipment, less toxic chemical and less sequencing runs than the BioBrick assembly. Second, it would make fast and efficient cloning accessible for both researchers as well as for less experimented users, such as high school iGEMers or biohackers thanks to the simplicity of its protocol. Third, the creation of large expression cassettes using this method is cheaper and faster than synthesizing the construct at the present day, which would guaranty the interest for a DNA database compared to <i>de-novo</i> synthesis for the years to come. And last, this method is a lot more easy to automatize unlike the RFC 10 biobrick format.</p><br />
<br />
<h1>Theory</h1><br />
<br />
<h2>Requirements for the development of the new standard</h2><br />
<br />
<p>In the continuation of what we said previously and in order to stay in the progression of the methodology we have tried in this work to stay as close as possible to biobrick format, to keep GoldenBrick parts compatible with the RFC 10 standard assembly, while opening new perspectives to increase the speed and the easiness of cloning. These constraints imposed several requirements for this new standard.</p><br />
<br />
<p>First, the new assembly method should:</p><br />
<ol><br />
<li>Keep the compatibility with the current BBF RFC 10 standard</li><br />
<li>Be compatible with a database approach</li><br />
<li>Minimize the number of different prefixes and suffixes</li><br />
<li>Use the standard registry plasmids and negative cloning control</li><br />
</ol><br />
<br />
<p>To improve the cloning speed, we would also like to:</p><br />
<ol start=5><br />
<li>Make one step Golden Gate cassette cloning possible</li><br />
<li>Allow to check the construct with a single enzyme digestion</li><br />
<li>Improve the compatibility with Gibson by removing the NotI site</li><br />
<li>Provide a solution for non standard assemblies</li><br />
</ol><br />
<br />
<p>As we will demonstrate, the GoldenBrick format we have established meet all these requirement. To understand on what basis we have created it, we are now going to explain in this section the principles that led the design of this format before we analyze the sequences of the GoldenBrick prefixes and suffixes and finally discuss of what can be done with this technique.<br />
<br />
<h2>A brief introduction on type II restriction enzymes</h2><br />
<br />
<p>Golden Gate cloning, (also known as type II restriction enzyme cloning) rely on a specific familly of enzyme (known as the type II enzymes) very different from the ones we are used to. The characteristic of type II enzymes from the cloning point ov view is that these enzymes cut the DNA outside their recognition locus, no matter the sequence of the place where they cut. A very small subtype of them can be used for cloning purpose, since they generate overhangs on one side of the recognition site only. The ones used for Golden Gate and MoClo cloning are BsaI and BbsI.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:202px;"><a href="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" width="200" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/0/0b/S%C3%A9lection_164.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 2: The recognition pattern of BsaI and cutting site.</div></div></div></div><br />
<br />
<br /><br />
<br />
<p>The fact that the enzyme cuts outside its recognition site gives them two significant advantages:</p><br />
<ul><br />
<li>You can generate as many different overhang you want with a single enzyme within the same tube</li><br />
<li>You can engineer your fragment so that it cannot be cutted again after it has ligated.</li><br />
</ul><br />
<br />
<p>Thanks to this last point, it is possible to cut and ligate the DNA in the mean time within the same tube (BsaI works in the T4 ligase buffer), and do not require any DNA purification. For this reason, the ligation is acheived with a better yeild and with less mutation than with traditional type I enzymes cloning methods.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/9/90/S%C3%A9lection_165.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Figure 3: Principle of the Golden Gate cloning method.</div></div></div></div><br />
<br />
<p>Since the small cut BsaI fragment can re-ligate with its mother strand, in order to improve the yield, the assembly is a lot more efficient when using a cycling between digestion and ligation phases in a thermocycler alternating between 37°C and 16°C, which drives the equilibrium towards the formation of the product.</p><br />
<br />
<h2>Analysis of a classical synthetic biology constructions</h2><br />
<br />
<p>A traditional construct in synthetic biology is made of the repetition of the following elements:</p> <br />
<br />
<br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:402px;"><a href="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" width="400" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/6/6e/Constructs.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 4: Schematic of the different elements assembly in a standard synthetic biology device.</div></div></div></div><br />
<br />
<p>At the moment, GoldenBrick only allow the assembly of a single cassette at the time. In order to create the full system, the assembly of the different cassettes together have to be conducted afterwards with the biobrick assembly, Gibson assembly - that is very efficient to assemble long fragments - or as we will discuss, ligation independant cloning []. Focusing closer on a single cassette, we can notice that we have 4 different kinds of junctions:</p><br />
<br />
<br /><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/2/26/Constructs_anotated.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 5: In a single cassette, we can notice 4 different kinds of junctions. If we want the repetition of the {RBD-CDS} unit to be possible, the prefix of the RBS have to be compatible with the suffix of the gene.</div></div></div></div><br />
<br />
<p>As we can notice on this picture, there is 4 different kinds of junctions in a casette of device. In Golden Gate, the junctions have the form of a DNA overhang that we can freely engineer thanks to the capacity of Type II enzymes to cut outside its regognition site, no matter what sequence is there. Therefore, we should engineer 4 different overhangs. The first overhang is dedicated to the ligation of the plasmid suffix with the promoter prefix (OH1). Similarely, the 5th one is dedicated to ligate the terminator suffix with the backbone prefix (OH5). In a bacterial device, the scar between the RBS and the gene (OH3) has to be very well controlled because the distance between the two elements is critical for correct protein expression. This scar will remain the same than in the biobrick assembly, but it will be generated by a type II enzyme as for the others overhangs. Finally, the overhang that link the RBS prefix to the promoter suffix (OH2) and the protein suffix to the terminator prefix (OH4) have to be compatible to make possible the repetition of several {RBS-Protein} units in a single cassette (OH2=OH4). We will see later how to control this repetition.</p><br />
<br />
<p>To conclude, the necessity for 4 different overhangs implies to create 5 different prefixes and 5 different suffixes depending on the function of the DNA elements.</p><br />
<br />
<p>In addition, we would like the scar generated by the assembly of all the different elements to be digestible by a single enzyme. This would enable to control whether all the elements we intended to insert in the construct has been assembled before sequencing the casette. As we will see later this element will be critical for the selection of the clone that have inserted all the genes we want. This is why the overhangs 1, 2, 4 and 5 has been engineered in the middle of a BbsI site. When two pieces ligates, they recreate a BbsI restriction site, and we can check the correct insertion of a given gene in the construct by simply digesting with BbsI and checking for the size on a gel.</p><br />
<br />
<p>One last condition to fulfil to get a RFC 10 compatible brick after GoldenBrick cloning is to leave no illegal restriction site inside the brick after the assembly of the cassette. However, we should keep the as similar as possible the biobrick extensions around each elements leaving the 4 usuals enzyme restriction sites, so that the brick can be still assembled using biobrick method. The only drawback we didn't managed to overcome is that if several GoldenBrick parts are assembled with RFC 10 standard, they cannot be assembled together using GoldenGate afterwards, because the biobrick assembly will leave illegal BsaI site inside the casette.</p><br />
<br />
<h2>The proposed new set of Golden Bricks extension</h2><br />
<br />
<p>Having all these design principles in mind and meeting the requirements we have proposed, we designed the new GoldenBrick prefix and suffix with the following sequences:</p><br />
<br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:635px;"><a href="/File:GoldenBricks.png" class="image"><img alt="" src="/wiki/images/e/ed/GoldenBricks.png" width="633" height="286" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GoldenBricks.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Figure 6: Sequence and design of the different prefixes and suffixes used in the GoldenGate cloning method. The presence of the EcoRI, XbaI, SpeI and PstI keep the compatibility with Biobrick cloning while the BsaI site enable single shot GoldenGate assembly. The BbsI sites serve as a single digestion control.</div></div></div></div><br />
<br />
<p>This design fulfil all the requirements enumerated before and ensure that the different elements will be assembled in the correct order. We also generate a scar that can be digested afterwards with the Bbsi enzyme, so that the correct insertion of the different elements can be check afterwards by doing a single digestion. This also enable the polymerization of the {RBS-CDS} units, as we will discuss in the next paragraph.</p><br />
<br />
<h2>Control over the polymerization</h2><br />
<br />
<p>The only difference with a standard GoldenBrick protocol is the capacity for the RBS-protein cassette to polymerize. This polymerization capacity makes the power of the technique because it enable to make polysistronic mRNAs no matter what gene is inserted. However, a control have to be achieved oved it in order to control when to create polysistrons and when we want a single gene to be inserted.</p><br />
<br />
<p>In order to acheive such a control, several parameters in the protocol will move the balance from mono-systrons to poly-sistrons. The first and the most obvious one is the stoechimoetry of the different elements. If the promoter and terminators are in large excess compared to the RBSs and the gene, the balance will be in favour of mono-sistrons. On the contrary, if the amount or RBSs and gene in dominant, the cassette will tends to contain two or more genes inserted inside.</p><br />
<br />
<p>On the other hand, the number of cycle and the ligation time will also influence the polymerization. Poly-sistrons are not likely to appear when the number of cycle is small and the ligation time short, but they will become more and more important as we increase these parameters.</p><br />
<br />
<p>Most of the work on GoldenBrick by our team is now focused in optimizing the protocol. We will release soon a set of standard protocols for mono and polysistrons optimized to reduce the polydispersity of the polymerization lenght.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="/File:GoldenBrick-polymerization.png" class="image"><img alt="" src="/wiki/images/1/10/GoldenBrick-polymerization.png" width="500" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GoldenBrick-polymerization.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 8: a. The vector, promoter, and terminator are in excess: the assembly produces mostly monosistrons. b. The protein sequences and RBS are in excess: the assembly produces mostly polysistrons.</div></div></div></div><br />
<br />
<br />
<h2>Assembly procedure</h2><br />
<br />
<div class="thumb tright"><div class="thumbinner" style="width:302px;"><a href="/File:GOldenbrick_global_thumb.png" class="image"><img alt="" src="/wiki/images/thumb/f/f3/GOldenbrick_global_thumb.png/300px-GOldenbrick_global_thumb.png" width="300" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="/File:GOldenbrick_global_thumb.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 9: Summary of the GoldenBrick procedure</div></div></div><br />
<br />
<br />
<p>Assembling parts using the GoldenBrick method is a lot easier than for BioBricks and also 5 to 10 times faster. We are now going to explain the procedure from the theoretical point of view. For the complete protocol, you should refers to the material and method section.</p><br />
<br />
<p>Because it requires a very small amount of DNA, the plasmid source can be taken directely from a DNA distribution suck as the partsregistry distribution, without requiring the need of a DNA preparation before. Once the plasmid are resuspended, the experimentalist mix them together in a mix containing the BsaI enzyme and T4 ligase and put them in the thermocycler for 5 hours. In the end, the product can be directely transformed and plated. A first round of screening sor the good polymerization length can be made using colony PCR with the standard VF2 and VR primers. If required, a second step of screening can be made after minipreping using BbsI. Given the very small rate of mutation, only two clones with the good digestion or colony PCR signature can be sent for sequencing; they will contain the correct construct with a very high probability.</p><br />
<br />
<p>This technique however requires to screen an important number of clones because we don't have a total control of what is assembling inside the cassette, because of the freedom we left for the RBS-Protein entities to polymerize. We will show in the next paragraph how we can gain a relative control over it. To reduce the costs of screening, we encourage the use of colony PCR oven mini-preping and digestion when possible.</p><br />
<br />
<br />
<h1>Other advantage of GoldenBricks over any other standard cloning techniques</h1><br />
<br />
<h2>Non standard assembly and "split-construct" vectors</h2><br />
<br />
<p>If you are willing ot create a non standard assembly or to control very precisely RBS-protein pairing over a polysistrons, you might like to create the construct in two times and assemble them together in a second step. This is why we have created the "split-construct" vectors. The split-contruct vectors are like the goldenbrick vectors except that they contain the OH2-OH5 or the OH1-OH4 overhangs instead of the OH1-OH5 overhangs as the GoldenBrick plasmid.</p><br />
<br />
<p>These vectors enable the assembly of shorter casette containing only a promoter, and a serie of RBS-protein for the "split-construct" 1 vector, or RBS-protein terminator for the "split-construct" 2 vector. This way, if a non standard biobrick assembly have to be carried in between the two pieces or if a long polysistron have to be created, they can be assembled using the standard EcoRI, XbaI, SpeI, PstI cloning, but several cloning step would have been saved by using GoldenBricks compared to a standard assembly.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/1/19/Split-constructs.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 10: Description of the construction of a non classical cassette for creating a device that test the strength of a terminator using split-vector GoldenBricked mixed with classical or Gibson assembly. The GFP and RFP rations are measure to evaluate the strengh of a terminator the classical way. Two cloning steps are avoided using this procedure.</div></div></div></div><br />
<br />
<br />
<h2>GoldenBrick make standard part DNA shuffling possible</h2><br />
<br />
<p>Aside from the dramatic shortening of the cloning time, the decrease of the costs and the easiness of GoldenBricks over Biobricks, one of the most noticeable improvement is undoubtedly the possibility to make DNA shuffling using a standard format. Lets take the example of a complex system such as a toogle-switch, in which you would like to optimize the expression of the protein repression of its two component to place the system in its working domain.</p><br />
<br />
<p>Using the Biobrick format, you will probably have to make tens of different constructs with different RBS to kind a condition in which the toogle-switch will work. Using GoldenBricks, you can make all of them in a single tube, in a single step. Moreover, you may eventually find a way to screen them directely after the cloning avoiding the need of purifying and sequence all the clones that couldn't work. The figure 11 shows in theory how one can proceed to such experiment design.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/3/31/DNA-shuffeling.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 11: Description of a DNA shuffling experiment in order to optimize the expression of a single gene. 4 Promoters and RBSs are mixed together in a single tube and give rise to 16 construct in a single tube in a single step. The clone expression the enzyme at the correct level can be screened directly on the transformation plate.</div></div></div></div><br />
<br />
<p>Many other use of DNA shuffling can be find in the literature especially for gene expression optimization and screening for working enzymes. We expect that DNA shuffling over standard parts is going to find many applications in the future of iGEM projects.</p><br />
<br />
<h2>GoldenBrick is still compatible with Biobrick format</h2><br />
<br />
<p>If the situation requires the part to be assembled with the standard RFC 10 format, the new GoldenBrick parts can be assembled the very same way than BioBricks. The only limitation is that BioBrick assembly of GoldenBrick parts leave a scar containing the BsaI restriction site inside, and the produced parts cannot be reassembled with other parts using Golden Gate. No simple way of fixing this issue has been found.</p><br />
<br />
<h2>GoldenBricked plasmids are compatible with standard Golden Gate assembly</h2><br />
<br />
<p>The GoldenBricked plasmids can be used to assemble contructions using classical Golden Gate. This option has been tested in our team and it works fine. (result not presented here)</p> <br />
<br />
<h2>GoldenBrick also assemble PCR products and shorten the assembly time</h2><br />
<br />
<p>Someone than want to create a construct not having the time or the need of cloning his gene into a vector first can stil assemble them using GoldenBrick. Although this pracice wouldn't be encouraged for standards parts, it can become really usefull when trying libraries of different genes in order to find a working one. GoldenBricked parts in plasmid and PCR GoldenBricked parts can be mixed together and assembled the very same way. The user will just have to take care to submit his working parts afterwards. This is why we recommend to amplify new parts flanked by its GoldenBricks prefix and suffix, mix them with the GoldenGate assembly mix in one hand, and digest them in EcoRI and PstI on the other in order to ligate them in pSB1C3 forming the GoldenBrick.<br />
<br />
<h2>Classical GoldenGate can be mixed GoldenBrick assembly to create fusion proteins</h2><br />
<br />
<p>GoldenGate in general and GoldenBricks in particular are very efficient to assemble fusion proteins, because one can design every overhang he wants and therefore fuse proteins without making any scar. New fusion proteins can be created while beeing assembled in the standard GoldenBrick technique with custom made scar designed by the experimentalist. A new fusion protein made of several domains can be assembled in the middle of a polysistron without having to make the fusion protein first before cloning.</p><br />
<br />
<h2>Finally, GoldenBricks works with eukaryotes chassis as well as with prokaryotes</h2><br />
<br />
<p>As we have seen all along this article, this design of GoldenBricks have been design for procaryotes, but it can work very well for eukaryotes. In eukaryotes, promoters can include or not a 5'UTR in their sequence to enhance the protein expression. In the case there is no 5'UTR, we can use the same prefix and suffix than the prokaryote promoters and include the 5'UTR with the prokaryotes prefix and suffix.</p><br />
<br />
<p>In order to make polysistrons, we can use an Intermediate Ribosome Entry Site (IRES), cloned between an prokaryote RBS prefix and suffix.</p><br />
<br />
<p>Finally, the polyA and the polyA capping cassette with a polymerase terminator can be flanked by the prokaryotes terminators sites. In the case one don't want to include a polyA capping, he can use the "split construct" 1 or in the case there is not terminator, the one on the plasmid will take care of stopping the RNA polymerase extension.</p><br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:802px;"><a href="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" width="800" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/0/0a/GoldenBricks-eukaryote.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 12: GoldenBricks sites for eukaryotes constructions.</div></div></div></div><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of the library</h2><br />
<br />
<p>In order to test the GoldenBrick technique efficiently, and for easiness of screening and making statistics, we have developed a set of GoldenBricked parts (see Material & Methods) with fluorescent proteins as a screening set. The parts have that we have created successfully are the following:<br />
<br />
<ul><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812050">K812050:</a> A GoldenBricked version of pSB1C3 with J04450 as negative cloning control (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812051">K812051:</a> A GoldenBricked version of pSB1K3 with J04450 as negative cloning control (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812053">K812053:</a> A GoldenBricked version of the strong RBS B0034 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812054">K812054:</a> A GoldenBricked version of the RFP E1010 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812055">K812055:</a> A GoldenBricked version of the terminator B0015 (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812056">K812056:</a> A GoldenBricked version of the pLac R0010 promoter (status=sent)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812057">K812057:</a> A GoldenBricked of an sfGFP protein (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812058:</a> A GoldenBricked of medium strenght RBS J61107 (status=constructed)</li><br />
<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812059:</a> A GoldenBricked of week RBS J61117 (status=constructed)</li><br />
</ul><br />
<br />
<p>All the parts written here have been successfully constructed and sequenced and the ones marked as "sent" has been sent to the PartsRegistry.</p><br />
<br />
<h2>Creation of a simple cassette</h2><br />
<br />
<p>We have started experimenting on the GoldenBrick assembly method. For the moment, the results are not optimal so we are not publishing them yet. Betters results are to come since we are optimizing the protocol now.</p><br />
<br />
<h2>Working with regular GoldenGate using GoldenBricks vectors</h2> <br />
<br />
<p>In our way of setting up new protocols and in order to assemble the fusion proteins we needed for our auxin system, we carried out two assemblies of fusion proteins using Golden Gate in our Golden Gated vectors. This paragraph reports the construction of the IaaH-Pep2A-IaaM fusion protein in the GoldenBrick plasmid K812050 using standard Golden Gate protocol. This protocol hadn't been optimized at the moment. However, out of 16 cloned on which we did colony PCR, 10 of them had the good size and out of the 3 sequenced, they were all correct.</p><br />
<br />
<br />
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:702px;"><a href="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" width="700" class="thumbimage" /></a> <div class="thumbcaption"><div class="magnify"><a href="https://static.igem.org/mediawiki/2012/thumb/4/40/IaaH-Pep2A-IaaM.png/800px-IaaH-Pep2A-IaaM.png" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 13: Colony PCR of 15 clones from the transformation plate. The ones marked with a star has been sequenced and were corrects. The one with and exclamation mark was sequenced and had only one of the CDS inserted.</div></div></div></div><br />
<br />
<p>This experiment demonstrates the possibility of doing Golden Gate in the GoldenBricks plasmids K812050 (and similarly K812051).</p><br />
<br />
<br />
<h1>Material and methods</h1><br />
<br />
<h2>Construction of the library</h2><br />
<br />
<p>The GoldenBricked parts were constructed amplifying the corresponding biobricks using primers flanked by the appropriate GoldenBricks extensions, using Phusion (Thermo Scientific) or Q5 (NEB) DNA polymerase in a 30 cycle reaction. The PCR product was PCR purified and then digested using FastDigest® EcoRI and PstI (Thermo Scientific). The standard plasmid pSB1C3 (or pSB1K3) were digested similarely. Both vectors and inserts were PCR purified and then ligated using T4 DNA ligase (Thermo Scientific) before being transformed with home made competent cells (chemical or electroporation).<p><br />
<br />
<p>The either red or non red colonies were minipreped and then digested with BsaI for screening. All the clones that had the correct digestion pattern were sequenced and gave the correct sequence.</p><br />
<br />
<p>The RBSs were synthetized in the form of two mathing oligos. A mixture of 10 µM of the two oligo was incubated at 98°C for 2 min and went down to 4°C with a ramp of -1°C/s. They were then PCR purified, digested and PCR purified again before beeing ligated in the same way.</p><br />
<br />
<h2>Assembly protocol</h2><br />
<br />
<p>The protocol given here han't been optimized yet. We are only starting the development of this new technology. It is given as a matter of indication. Some optimized protocol will be available soon.</p><br />
<br />
<p>About 75 ng of each plasmid (more accurate ratios will be optimized) were mixed together with 2.5 units of BsaI enzyme (NEB) and 15 units of T4 DNA ligase (Thermo Scientific) in a total volume of 15 µL of standard T4 ligation buffer. The tubes were then placed in the thermocycler with with the following steps:</p><br />
<br />
<ol><br />
<li>37°C, 2 min (digestion)</li><br />
<li>16°C, 5 min (ligation)</li><br />
<li>Goto 1, 50 times</li><br />
<li><ul><li>Option 1: if the construct does not contain any BsaI site: 50°C 5 min (reduce the background with a final digestion)</li><br />
<li>Option 2: if the construct contains BsaI site: 16°C 2h (reconstitute the BsaI site)</li></ul></li><br />
<li>80°C, 5 min (heat inactivation)</li><br />
</ol><br />
<br />
<p>5 µL of the product was transformed with 50 µL of home made chemically competent cells and recovered for 1 hour. The cells were concentrated and plated.</p><br />
<br />
<h1>Perspectives for GoldenBricks and the PartsRegistry</h1><br />
<br />
<p>When the complete procedure will be established, our team will submit an BBF RFC request for the registry. If the RFC get accepted and if the tenant of the PartsRegistry thinks this is a valuable format, we can think of migrating the registry to this new format. This can be done in two ways: the first one is to ask every user to submit their new part in the new GoldeBrick format, and the second is to take advantage of the huge community working on the PartsRegistry and especially the iGEM comptetion. In order to migrate the parts faster, we can add in the requirements for the one of the medals for the teams to resubmit already exsiting parts in the new format. With an average of 200 teams in the years to come and asking each of them to resubmit 2 goldenbricked parts, we can cover the 2000 most useful parts within 2 or 3 years, because the teams will goldenbrick the parts they are interested in for their assembly, so they will probably always re-submit more than 2.</p><br />
<br />
<p>We beleive that implementing such a method in the partsregistry will speed up the assembly process and give a new shine to this database that severely suffers from the gene synthesis and PCR based methods competition. This will leave more time for researchers and iGEMers for characterizing the parts, and the partregistry will also benefits from an increase of parts characterization, which is very beneficial to the community. Overhall, the idea of standardized parts that tends to loose its strengh with the time will gain again in interest in the long run.</p> <br />
<br />
<h1>Conclusion</h1><br />
<br />
<p>Our team this year have created a new cloning format for the PartsRegistry that dramatically speed up the cloning time and open new perspective such as simple protein fusion methods and DNA shuffling that were not possible using the current BioBrick format. At the moment, we are working very hard to demonstrate its possibilities and come up with an efficient and reliable protocol. If the process is found to be efficient, we will submit an RFC request to the PartsRegistry and propose that GoldenBrick progressively substitute to the current Biobrick format, which will renew the interest for people on standardized biological parts, seriously challenged by gene synthesis and PCR based methods today.</p><br />
<br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>An introduction to agent-based modeling: Modeling natural, social and engineered complex systems with NetLogo</i>, Wilensky, U., & Rand, W. (in press), Cambridge, MA: MIT Press</li><br />
</ol><br />
</div><br />
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<center><h1>Intertissue communication: An orthogonal hormonal system</h1></center><br />
<h4><p>We adapted the auxin production device from the iGEM team Imperial college 2007 to eucaryotes and combined it with an auxin detection module. This way, we created the first synthetic hormonal system for intertissues communication.</p></h4><br />
<p>To test this system, we <a href="https://2012.igem.org/Team:Evry/InjectionTuto">co-injected</a> plasmids expressing our production and reception devices in tadpole's embryos, <a href="https://2012.igem.org/Team:Evry/FrenchFrog">a new chassis</a> we wanted to implement for synthetic biology. We performed auxin <a href="https://2012.igem.org/Team:Evry/AuxinTOX">toxicity</a> and <a href="https://2012.igem.org/Team:Evry/auxin_uptake">uptake</a> tests at the begining of our project to ensure the feasability.</p><br />
<br />
<h2>Auxin production devices</h2><br />
<p>We envisaged 3 ways of design our auxin production device in tadpoles. The devices 1 and 2 have been designed to be expressed in tadpole's embryos, while device 3 have been designed to be expressed in E.coli. In this last case, the aim is that <a href="https://2012.igem.org/Team:Evry/BXcom">the tadpoles eat bacteria</a> expressing device 3.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/Prodrecep.jpg" width="600px" alt="3 devices for production" /><br />
</center><br />
<ul><br />
<li> <b>Auxin production device 1 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812121">BBa_K812021</a>, coding for IaaM, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812120">BBa_K812120</a>, coding for IaaH for auxin generator for the use in tadpole's embryos.<br />
<br />
<li> <b>Auxin production device 2 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812014">BBa_K812014</a>. It is ment for the co-expression of IaaH and IaaM genes in the same cell in tadpole. <br />
<li> <b>Auxin production device 3 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515100">BBa_K515100</a>, coding for IaaM and IaaH for auxin generator in <i>E.coli</i>.<br />
</ul><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/b/bd/ProductiondeviceCompress.jpg" width="900px" alt="Production devices" /><br />
</center><br />
<br />
<h3>Pathway</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Pathway2.jpg" width="600px" alt="Auxin pathway" /><br />
</center><br />
<br />
<h2>Auxin reception devices</h2><br />
<p>We envisaged 2 ways of design our auxin production device in tadpoles. To visualize the communication between different tissues or between E.coli and a tissue of the tadpole, we chose to work with GFP.Our orthogonal hormonal system <b>works with any proteins fused to AID signal</b> with any transcription factors.</p><br />
<ul><br />
<li> <b>Auxin reception device 1 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a> coding for GFP-AID, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812012">BBa_K812012</a> coding for OsTir1.<br />
<li> <b>Auxin reception device 2 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812013">BBa_K812013</a> coding for GFP-AID and OsTir1 in the same cell.<br />
</ul></br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/ReceptionCompress.jpg" width="930px" alt="devices for reception" /><br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>1. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature methods 6, 917-22 (2009).</li><br />
<br />
<br />
</ol><br />
</div><br />
<br><br />
<br />
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<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<h1>Intertissue communication: An orthogonal hormonal system</h1><br />
<h4><p>We adapted the auxin production device from the iGEM team Imperial college 2007 to eucaryotes and combined it with an auxin detection module. This way, we created the first synthetic hormonal system for intertissues communication.</p></h4><br />
<p>To test this system, we <a href="https://2012.igem.org/Team:Evry/InjectionTuto">co-injected</a> plasmids expressing our production and reception devices in tadpole's embryos, <a href="https://2012.igem.org/Team:Evry/FrenchFrog">a new chassis</a> we wanted to implement for synthetic biology. We performed auxin <a href="https://2012.igem.org/Team:Evry/AuxinTOX">toxicity</a> and <a href="https://2012.igem.org/Team:Evry/auxin_uptake">uptake</a> tests at the begining of our project to ensure the feasability.</p><br />
<br />
<h2>Auxin production devices</h2><br />
<p>We envisaged 3 ways of design our auxin production device in tadpoles. The devices 1 and 2 have been designed to be expressed in tadpole's embryos, while device 3 have been designed to be expressed in E.coli. In this last case, the aim is that <a href="https://2012.igem.org/Team:Evry/BXcom">the tadpoles eat bacteria</a> expressing device 3.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/Prodrecep.jpg" width="600px" alt="3 devices for production" /><br />
</center><br />
<ul><br />
<li> <b>Auxin production device 1 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812121">BBa_K812021</a>, coding for IaaM, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812120">BBa_K812120</a>, coding for IaaH for auxin generator for the use in tadpole's embryos.<br />
<br />
<li> <b>Auxin production device 2 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812014">BBa_K812014</a>. It is ment for the co-expression of IaaH and IaaM genes in the same cell in tadpole. <br />
<li> <b>Auxin production device 3 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515100">BBa_K515100</a>, coding for IaaM and IaaH for auxin generator in <i>E.coli</i>.<br />
</ul><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/b/bd/ProductiondeviceCompress.jpg" width="900px" alt="Production devices" /><br />
</center><br />
<br />
<h3>Pathway</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/4/4b/Pathway2.jpg" width="600px" alt="Auxin pathway" /><br />
</center><br />
<br />
<h2>Auxin reception devices</h2><br />
<p>We envisaged 2 ways of design our auxin production device in tadpoles. To visualize the communication between different tissues or between E.coli and a tissue of the tadpole, we chose to work with GFP.Our orthogonal hormonal system <b>works with any proteins fused to AID signal</b> with any transcription factors.</p><br />
<ul><br />
<li> <b>Auxin reception device 1 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a> coding for GFP-AID, and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812012">BBa_K812012</a> coding for OsTir1.<br />
<li> <b>Auxin reception device 2 :</b> this device is composed of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812013">BBa_K812013</a> coding for GFP-AID and OsTir1 in the same cell.<br />
</ul></br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f2/ReceptionCompress.jpg" width="930px" alt="devices for reception" /><br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>1. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature methods 6, 917-22 (2009).</li><br />
<br />
<br />
</ol><br />
</div><br />
<br><br />
<br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:47:02Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>GoldenBrick: A new biobrick cloning format</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling a tadpole: A multi-level approach</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone">Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick">GoldenBrick: A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling">Modeling a tadpole: A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human">Human practice: What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
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<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:41:50Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Intertissue communication: An orthogonal hormonal system </li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new chassis for multicellular synthetic biology</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone"><b></b>Intertissue communication: An orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling"><b>Modeling a tadpole:</b> A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:38:27Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis for synthetic biology</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Engineering a synthetic, orthogonal hormone as a communication device</li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new chassis for multicellular synthetic biology</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone"><b></b>A synthetic, orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling"><b>Modeling a tadpole:</b> A multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
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<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:33:56Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis for synthetic biology</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Engineering a synthetic, orthogonal hormone as a communication device</li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new chassis for multicellular synthetic biology</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone"><b></b>A synthetic, orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling"><b>Modeling a tadpole:</b> a multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
<br />
<!--<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 300px; height: 50px; text-align: center; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<br /><br />
<p><a href="https://2012.igem.org/Team:Evry/Modeling">Visit the project page ></a></p><br />
<br /><br />
</font><br />
</div><br />
<br />
<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
<p><a href="https://2012.igem.org/Team:Evry/BGame">Visit the project page ></a></p><br />
!--><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/ProjectTeam:Evry/Project2012-09-25T20:32:53Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis for synthetic biology</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Engineering a synthetic, orthogonal hormone as a communication device</li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new chassis for multicellular synthetic biology</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" target="_blank" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone"><b></b>A synthetic, orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
<br />
<!-- GoldenBrick --><br />
<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
<br />
<!-- Modeling --><br />
<h2 id="modeling"><b>Modeling a tadpole:</b> a multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
<!-- Human Practice --><br />
<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
<br><br><br />
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<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
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</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-25T20:29:23Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><h1><b><i>Xenopus tropicalis</i>: A new multicellular chassis</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> <br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and inter-tissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multi cellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <a href="https://2012.igem.org/Team:Evry/AIDSystem">A synthetic, orthogonal hormonal system.</a> </p><br/><br />
<br />
<br />
<h2>The simple molecular strategy to build eukaryotic plasmid ready to use: </h2><br />
<br/><br/><br/><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br />
<br/><br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in Xenopus</h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage 48-50</a>. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>We injected 2.3 nl of plasmids at 100ng.µl-1 - embryos were stored at 21°C during all the experiment. pCS2+ GFP-aid: contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
<br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>We showed that our constructions express our reporters with the CMV promoter. These parts are considered characterized. Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Spectral variants of fluorescent proteins could be expressed in different tissues. Within one tadpole the fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.</P><br />
<P>Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.</P><br />
<P>Moreover the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell.</p><br><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-25T20:29:02Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><h1><b><i>Xenopus tropicalis</i>: A new chassis for multicellular synthetic biology</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> <br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and inter-tissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multi cellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <a href="https://2012.igem.org/Team:Evry/AIDSystem">A synthetic, orthogonal hormonal system.</a> </p><br/><br />
<br />
<br />
<h2>The simple molecular strategy to build eukaryotic plasmid ready to use: </h2><br />
<br/><br/><br/><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br />
<br/><br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in Xenopus</h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage 48-50</a>. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>We injected 2.3 nl of plasmids at 100ng.µl-1 - embryos were stored at 21°C during all the experiment. pCS2+ GFP-aid: contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
<br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>We showed that our constructions express our reporters with the CMV promoter. These parts are considered characterized. Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Spectral variants of fluorescent proteins could be expressed in different tissues. Within one tadpole the fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.</P><br />
<P>Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.</P><br />
<P>Moreover the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell.</p><br><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-25T20:28:16Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<html><br />
<center><h1><b>Xenopus tropicalis: A new chassis for multicellular synthetic biology</b></h1></center><br />
<br />
<h2>Establishment of a new chassis</b></h2><br/><br />
<br />
<p>So far, synthetic biology has mostly focused on bacteria, since they are simple to engineer. iGEM teams and laboratories have worked on unicellular organisms in order to understand the underlying biology and have developed an impressive database of molecular parts. Some work has also been done on engineering mammalian cells and a few iGEM teams have followed this trend. Synthetic biologists are now imagining the rational design of multicellular organisms with numerous applications ranging from gene therapy or drug production to environmental monitoring. This year, our team would like to be part of that challenge.</p><br />
<br />
<p>The arrival of <i>Xenopus</i> as a chassis in synthetic biology requires the creation of new standards and protocols that the community will be able to build on. We provided the registry with such tools that allow rapid construction and characterization of devices in vivo, and include debugging tools. We think they will be very useful for later iGEM teams and synthetic biologists who wish to work with <i>Xenopus</i> for building multicellular systems.</p><br />
<br />
<p>You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to <i>Xenopus</i> page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.</p> <br />
<br />
<p>This year, the Evry iGEM team is going to be the one of the first iGEM team to work on a vertebrate. Our work is focused both on developing a system for intercellular and inter-tissue communication, and creating the tools for the iGEM community to easily express genes in specific tissues. We believe the tadpole is a chassis of choice for iGEM on multi cellular organisms, as experiments can be conducted in one week using microinjection methods. We hope to demonstrate the feasibility of engineering <i> Xenopus </i> in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: <a href="https://2012.igem.org/Team:Evry/AIDSystem">A synthetic, orthogonal hormonal system.</a> </p><br/><br />
<br />
<br />
<h2>The simple molecular strategy to build eukaryotic plasmid ready to use: </h2><br />
<br/><br/><br/><br />
<a href="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0e/French_froggies_scheme2.1.png" alt="Image unavailable" width="950px" /> </a><br />
<br/><br/><br/><br/><br />
<br />
<br />
<h2>Example of GFP expression in Xenopus</h2><br/><br />
<br />
<p>The <a href="https://2012.igem.org/Team:Evry/InjectionTuto">injection tutorial</a> explains very simply with diagram how we did injection and how take care about your embryos and tadpole. The experiment carries on 5 days, from the unfertilized egg to a swimming tadpole at <a href="https://2012.igem.org/Team:Evry/Stages">stage 48-50</a>. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).<br />
<br />
<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/><br />
<br />
<p>We injected 2.3 nl of plasmids at 100ng.µl-1 - embryos were stored at 21°C during all the experiment. pCS2+ GFP-aid: contains the constitutive and ubiquitous promoter CMV and the aid sequenced of the aid system fusionned to GFP (Green Fluorescent Protein)(Nishimura et al., 2009), this Biobrick created by our team is <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">BBa_K812010</a>, and it was integrated into our Eucaryotic plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>.We injected about 3.78E+7 plasmids.</p><br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Tadpole_de_la_mort.png" alt="Image unavailable" width="950px" /> <br />
<br />
<h4><b>The characterization of all reporter and promoters is <u><a href="https://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br><br />
<br />
<h2>Conclusion</h2><br/><br />
<br />
<p>We showed that our constructions express our reporters with the CMV promoter. These parts are considered characterized. Nevertheless we expected a more uniform expression of the reporters with the CMV promoter. Spectral variants of fluorescent proteins could be expressed in different tissues. Within one tadpole the fluorescent proteins were observed in one to four different tissues, and tissues were different between tadpoles.</P><br />
<P>Explanation: The plasmid DNA does not diffuse in the egg and stay in the same area around the injection site. This means that depending on the injection site, the plasmid will be inherited by a given set of cells within the tadpole. This question was raised in our <a href="https://2012.igem.org/Team:Evry/plasmid_splitting">model</a>. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.</P><br />
<P>Moreover the expression of reporters decreases during time, because plasmid DNA is subjected to a catabolic activity during development but also plasmid DNA gets diluted as cells proliferate and the quantity of plasmid DNA decreases for each cell.</p><br><br />
<br />
<br />
<div id="citation_box"><br />
<p id="references">References:</p><br />
<ol><br />
<li><i>Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines.</i>, Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002</li><br />
<br />
<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li><br />
<br />
<li><i>An auxin-based degron system for the rapid depletion of proteins in nonplant cells</i>, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009</li><br />
</ol><br />
</div><br />
<br><br />
<br />
<script type="text/javascript">writeFooter()</script> <br />
</html></div>Tiffhttp://2012.igem.org/Team:Evry/FrenchFrogTeam:Evry/FrenchFrog2012-09-25T20:26:55Z<p>Tiff: </p>
<hr />
<div>{{:Team:Evry/template_v1}}<br />
<br />
<!-- contents --><br />
<br />
<html><br />
<br />
<h1>Project overview</h1><br />
<p><br />
For our first participation in iGEM, we have decided to introduce a new organism to the competition: <i>Xenopus tropicalis</i>. Its common name is the Western clawed frog, a diploid cousin of the model organism <i>Xenopus laevis</i>. Aside for the soft spot us French have for frogs, we also believe <i>Xenopus</i> could be a great multicellular chassis for synthetic biology. We are therefore bringing this organism to iGEM for the first time, along with the tools we need to bring Synthetic biology to the multicellular era.<br />
</p><br />
<br />
<p><br />
The laboratory part of our work can be divided into two categories: the creation of synthetic biology tools for <i>Xenopus</i> and the creation of a synthetic hormonal system. We created a multi-level model of this inter-tissue communication system, concurrently laying the groundwork for modeling of synthetic genetic systems in multicellular organisms. Finally, using vertebrates in synthetic biology poses deep ethical problems, which come alongside those of animal experimentation. iGEM aims to be cool and fun, but can we or should we keep the same attitude when working with vertebrate embryos ? Should we reduce animals to objects or tools by using words such as chassis when working with these multicellular organisms? Our resident philosopher lead our team’s reflection on these issues, proposing a guide for future synthetic biologists who wish to work with <i>Xenopus</i>. <br />
</p><br />
<br />
<div id="contourmenu" style="position: relative; margin-left: auto; margin-right: auto; width: 700px; text-align: left; background-color: #cee9f4; border-radius: 7px; align: left"><br />
<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br><br />
<div id="item" style="margin-left:20px;"><br />
<ul><br />
<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a><br />
<a href="#hormone" style="text-decoration:none;"><li>Engineering a synthetic, orthogonal hormone as a communication device</li></a><br />
<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a><br />
<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a><br />
<a href="#human" style="text-decoration:none;"><li>Human pratice: A philosophical investigation into the introduction of <i> Xenopus tropicalis </i> in iGEM </li></a><br><br />
</ul><br />
</div><br />
</font><br />
</div><br />
<br />
<br><br><br />
<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center><br />
<br><br><br />
<br />
<!-- Bannière Grenouille --><br />
<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center><br />
<!-- #Bannière Grenouille --><br />
<br />
<br><br><br><br />
<!-- Xenopus Tropicalis --><br />
<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2><br />
<br />
<p><br />
While iGEM has so far been mostly focused on en engineering unicellular organisms and bacteria in particular, we have decided work on the next step for synthetic biology: Multicellular engineering. Using our new biobrick tools, the road towards synthetic circuits in <i>Xenopus</i> is open, multiplying the number of potential applications in terms of synthetic biology.<br />
</p><br />
<br />
<p><br />
<i>Xenopus</i> is a model organism for developmental biology. Rapid development, easy handling and direct injection of plasmids into the fertilized egg allow testing of constructs in less than two weeks. We provide frog compatible plasmids for using this system in biobrick standard format. We have submitted two complementary systems: The first allows rapid testing of the system, but in a transient way. It also includes tools for debugging of the system. Once a working system is in place, it can be reassembled and integrated on the chromosome for longer-term use.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a><br />
<br />
<!-- AID System --><br />
<h2 id="hormone"><b></b>A synthetic, orthogonal hormonal system </h2><br />
<p><br />
In order to be able to bring synthetic biology to multicellular organisms, it is essential that we have a communication device enabling cell-to-cell or tissue-to-tissue communication. We have advanced towards the implementation of the first synthetic hormone, which would allow communication between cells in an orthogonal manner. We have submitted an auxin receiver device to the registry, for use in combination with an auxin production system that we have adapted for eukaryotic chassis. <br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/AIDSystem">More details here...</a><br />
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<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2><br />
<p><br />
We have started the development ou a new biobrick format that may revolutionize the future of iGEM cloning by enabling the assembly of a complete expression cassette in one shot, in a cheaper and most reliable way than all the current cloning method. We are the initiator of that project and we are going to develop it in partneship with the iGEM Paris Bettencourt and the CINVESTAV-IPN-UNAM_MX team in Mexico.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/GB">More details here...</a><br />
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<!-- Modeling --><br />
<h2 id="modeling"><b>Modeling a tadpole:</b> a multi-level approach</h2><br />
<p><br />
Modeling a synthetic system at the whole organism scale is a challenge in itself. Using differential equations and agent based simulation, we aimed at modeling our entire synthetic hormonal system, as well as providing a new set of mathematical tool for iGEM in order to model complete organisms.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/Modeling">More details here...</a><br />
<br />
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<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2><br />
<p><br />
Working with vertebrate embryos raised many questions among the team. We decided to track the changes in our attitude to animal experimentation during our project. Before starting our experiments, we answered a list of questions concerning animal experimentation and synthetic biology. Our reflection progressively lead us to realize that considering animals as mere chassis waiting to be engineered was quite a problematic conception that we had to question.<br />
</p><br />
<a class="moredetails" href="https://2012.igem.org/Team:Evry/HumanPractice">More details here...</a><br />
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<h2 id="design"> style="text-decoration:none; color: white;"><b>Biotic games:</b> Better understanding of tadpole behavior through gaming</a></h2><br />
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