Team:Evry/FrenchFrog

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<center><h1><b>Xenopus tropicalis: A new chassis for multicellular synthetic biology</b></h1></center>
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<h1>Project overview</h1>
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<h2>Establishment of a new chassis</b></h2><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 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.
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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 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>
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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>.  
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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>
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<h3 style="text-align: center; padding-top:10px;">The French Froggies Project</h3><br>
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<a href="#xenopus" style="text-decoration:none;"><li><i>Xenopus tropicalis</i>: A new multicellular chassis</li></a>
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<a href="#hormone" style="text-decoration:none;"><li>Engineering a synthetic, orthogonal hormone as a communication device</li></a>
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<a href="#goldenBrick" style="text-decoration:none;"><li>Goldenbrick: A new Biobrick standard for one-shot assembly of multiple parts</li></a>
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<a href="#modeling" style="text-decoration:none;"><li>Modeling: A multi-level approach to model synthetic systems in multicellular organisms</li></a>
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<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>
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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>  
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<center>A quick summary of each project is proposed in this page. You can find more details on their specific pages. </center>
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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 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="http://2012.igem.org/Team:Evry/AIDSystem">A synthetic, orthogonal hormonal system.</a> </p><br/>
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<center> <img src="http://image.noelshack.com/fichiers/2012/27/1341654981-gregre.gif" width=800 /> </center>
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<h2 id="xenopus"><i>Xenopus tropicalis</i>: A new multicellular chassis</h2>
 
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<h2>The simple molecular strategy to build eukaryotic plasmid ready to use: </h2>
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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.
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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.
 
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<a class="moredetails" href="http://2012.igem.org/Team:Evry/FrenchFrog">More details here...</a>
 
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<h2>Example of GFP expression in Xenopus</h2><br/>
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<h2 id="hormone"><b></b>A synthetic, orthogonal hormonal system </h2>
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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.
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<a class="moredetails" href="http://2012.igem.org/Team:Evry/AIDSystem">More details here...</a>
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<p>The <a href="http://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="http://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).
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<h2 id="goldenBrick"><b>GoldenBrick:</b> A new biobrick cloning format</h2>
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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.
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<a class="moredetails" href="http://2012.igem.org/Team:Evry/GB">More details here...</a>
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<h3>Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter</h3><br/>
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<h2 id="modeling"><b>Modeling a tadpole:</b> a multi-level approach</h2>
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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.
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<a class="moredetails" href="http://2012.igem.org/Team:Evry/Modeling">More details here...</a>
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<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/>
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<h2 id="human"><b>Human practice:</b> What does it mean to be a chassis ?</h2>
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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.
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<a class="moredetails" href="http://2012.igem.org/Team:Evry/HumanPractice">More details here...</a>
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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>
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<h4><b>The characterization of all reporter and promoters is <u><a href="http://2012.igem.org/Team:Evry/Tadpole_injection1">here</a></u>.</b></h4><br>
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      title='Recent changes'>Recent changes</a></li>
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<h2>Conclusion</h2><br/>
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<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>
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<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="http://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>
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<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>
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<p id="references">References:</p>
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<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>
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<li><i>Xenopus: a prince among models for pronephric kidney development.</i>, Jones E., JASN 16:2, 2005</li>
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<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>
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Revision as of 20:28, 25 September 2012

Xenopus tropicalis: A new chassis for multicellular synthetic biology

Establishment of a new chassis


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.

The arrival of Xenopus 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 Xenopus for building multicellular systems.

You want to make the move from bacteria to multicellular synthetic biology ? Make sure you check out our Introduction to Xenopus page, and our Frogs for dummies page to make sure you are aware of all the differences between genetic engineering in eukaryotes.

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 Xenopus in one summer for an iGEM project, and to create a great tool for multicellular synthetic biology: A synthetic, orthogonal hormonal system.


The simple molecular strategy to build eukaryotic plasmid ready to use:




Image unavailable



Example of GFP expression in Xenopus


The injection tutorial 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 stage 48-50. The GFP (or other fluorescent protein) is expressed few hours after the fertilization to the end of the week (see below).

Plasmid injected: pCS2+ with CMV promoter and GFP-aid reporter


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 BBa_K812010, and it was integrated into our Eucaryotic plasmid BBa_K812000.We injected about 3.78E+7 plasmids.


Image unavailable

The characterization of all reporter and promoters is here.


Conclusion


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.

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 model. An other reason could be that the metabolism of each tissue specific cell is different and change during the tadpole's development.

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.


References:

  1. Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines., Chae J., Zimmerman L.B., Grainger R.M., Mechanisms of development 117:1-2, 2002
  2. Xenopus: a prince among models for pronephric kidney development., Jones E., JASN 16:2, 2005
  3. An auxin-based degron system for the rapid depletion of proteins in nonplant cells, Nishimura K., Fukagawa T., Takisawa H., Kakimoto T., Kanemaki M., Nature Methods 6:12, 2009