Team:Evry/AIDSystem

From 2012.igem.org

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<h1>Orthogonal hormonal system</h1>
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<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>
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<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>
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<h1> The Auxin-inductible degron system </h1>
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<h2>Auxin production devices</h2>
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<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>
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<center>
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<img src="https://static.igem.org/mediawiki/2012/9/97/Prodrecep.jpg" width="600px" alt="3 devices for production" />
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</center>
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<ul>
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<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.
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<h2>Overview</h2>
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<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.
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<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>.
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<center>
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<img src="https://static.igem.org/mediawiki/2012/b/bd/ProductiondeviceCompress.jpg" width="900px" alt="Production devices" />
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</center>
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<h3>Pathway</h3>
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<img src="https://static.igem.org/mediawiki/2012/4/4b/Pathway2.jpg" width="600px" alt="Auxin pathway" />
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</center>
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<h2>Auxin reception devices</h2>
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<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>
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Auxin-inductible degron (AID) system is a new tool to regulate protein expression specifically through induction of the proteolysis system by auxin.</br>  
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<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.
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Auxin represents a family of plant hormones that control gene expression during many aspects of growth and development.</br>
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<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.
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Figure 1 show how this system works:</br>
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<img src="https://static.igem.org/mediawiki/2012/f/f2/ReceptionCompress.jpg" width="930px" alt="devices for reception" />
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<center><img src="https://static.igem.org/mediawiki/2012/8/8d/Aid_system.jpg"/></center>
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<center><u>Figure 1: Schematic illustration of the AID system</u></center>
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<center><u>Source:</u>http://www.cosmobio.co.jp/export_e/products/proteins_and_peptides/brs_20110824.asp?entry_id=7884</center>
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</br>
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Auxin family hormones, such as indole-3-acetic acid (IAA, a natural auxin) and 1-naphthaleneacetic acid (NAA, a synthetic auxin), bind to the F-box transport inhibitor response 1 (TIR1) protein and promote the interaction of the E3 ubiquitin ligase SCF-TIR1 (a form of SCF complex containing TIR1) and the auxin or IAA (AUX/IAA) transcription repressors. SCF-TIR1 recruits an E2 ubiquitin conjugating enzyme that then polyubiquitylates AUX/IAAs resulting in rapid degradation of the latter by the proteasome.</br>
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The AID plasmid is composed like it shows in this figure:</br>
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<p id="references">References:</p>
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<center><img src="https://static.igem.org/mediawiki/2012/f/fa/Aid_plasmid.jpg"/></center>
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<ol>
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<center><u>Figure 2: Schematic composition of AID plasmid</u></center>
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<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>
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<center><u>Source:</u>http://www.cosmobio.co.jp/export_e/products/proteins_and_peptides/brs_20110824.asp?entry_id=7884</center>
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</br>
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The great advantage is that you can bind a reporter like GFP to aid. That allows you to follow in live the degradation of the target protein:</br>
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<center><img src="https://static.igem.org/mediawiki/2012/3/3b/Aid_GFP.png"/></center>
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<center><u>Figure 3: Immunofluoresence analysis of protein depletion by the AID system</u></center>
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<center><u>Source:</u>“An auxin-based degron system for the rapid depletion of proteins in nonplant cells”, Nature Methods. 2009 Dec;6(12):917-22. Epub 2009 Nov 15</center>
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</br>
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This system can be used to induce rapid depletion of a protein of interest (within 30 min) in the presence of auxin, and the reaction was reversible and tunable. This system worked in budding yeast as well as in cell lines derived from chicken, mouse, hamster, monkey and human, implying that it could be applicable to most eukaryotes, except plant species.</br>
 
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NB: Thanks to the work of Nishimura and al. (“An auxin-based degron system for the rapid depletion of proteins in nonplant cells”, Nature Methods. 2009 Dec;6(12):917-22. Epub 2009 Nov 15) we introduce the AID system in iGem competition. This paper is a great source of knowledge for our project, that’s why a lot of information is extracted from it in this page.</br>
 
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<h2>Advantages</h2>
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</ol>
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AID system shows lot of advantages compared to other degradation based method to control protein expression:</br>
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<center><img src="https://static.igem.org/mediawiki/2012/1/15/Comparison_degron.png"/></center>
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<center><u>Figure 4: comparison of degradation based methods to control protein expression</u></center>
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<center><u>Source:</u>“An auxin-based degron system for the rapid depletion of proteins in nonplant cells”, Nature Methods. 2009 Dec;6(12):917-22. Epub 2009 Nov 15</center>
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Moreover, there is a great advantage of using auxin to control protein expression. Its molecule in only active in plants and appears to be relatively silent in nonplant eukaryotes. Because it’s a small molecule this system allowed us to degrade protein in cytoplasm but also in nucleus. Moreover, this molecule is cheap and 20-500 µM of auxin should be enough to induce maximum depletion for most animal cells. Another advantage is that the culture can be maintained at a constant temperature.</br>
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The only limitation of AID system is that it can’t be used to control endogenous proteins without manipulation of the gene of interest.</br>
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<h2>Applications</h2>
 
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With the AID system, we can imagine a lot of application in Synthetic Biology.</br>
 
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This system is very flexible, fast and reversible. Because you just have to introduce the plamid with you target protein, this system is very powerful for protein control.
 
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With our new chassis, the xenopus, AID system could be a new source of iGEM projects.
 
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</br>
 
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If you want to stop protein effect during a certain time, you just have to put your tadpoles in a auxin bath, it’s not harmful for them.</br>
 
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A great application in future could be the chastity belt.</br>
 
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Indeed, the question of genetically modified organism is very important in Synthetic biology. We’re still searching a response to avoid the dissemination of modified species in nature. With AID system we can imagine a way to avoid this problem.</br>
 
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In tadpoles there are genes responsible of sex determination (DM-W and DMRT1). With degron system is simple to avoid expression of these genes. Like this we can make only male or only female in our lab, avoiding the possibility of reproduction of our species.</br>
 
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Possibilities are vast with AID system but we haven’t the time to explore it. That’s why our work is very useful for iGEM community, we wish new team will work on the AID system and explore the possibilities it procure.</br>
 
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<center><b>It could be you the next student working on it!</b></center>
 
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Revision as of 20:51, 23 September 2012

Orthogonal hormonal system

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.

To test this system, we co-injected plasmids expressing our production and reception devices in tadpole's embryos, a new chassis we wanted to implement for synthetic biology. We performed auxin toxicity and uptake tests at the begining of our project to ensure the feasability.

Auxin production devices

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 the tadpoles eat bacteria expressing device 3.

3 devices for production
  • Auxin production device 1 : this device is composed of BBa_K812021, coding for IaaM, and BBa_K812120, coding for IaaH for auxin generator for the use in tadpole's embryos.
  • Auxin production device 2 : this device is composed of BBa_K812014. It is ment for the co-expression of IaaH and IaaM genes in the same cell in tadpole.
  • Auxin production device 3 : this device is composed of BBa_K515100, coding for IaaM and IaaH for auxin generator in E.coli.
Production devices

Pathway

Auxin pathway

Auxin reception devices

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 works with any proteins fused to AID signal with any transcription factors.

  • Auxin reception device 1 : this device is composed of BBa_K812010 coding for GFP-AID, and BBa_K812012 coding for OsTir1.
  • Auxin reception device 2 : this device is composed of BBa_K812013 coding for GFP-AID and OsTir1 in the same cell.

devices for reception

References:

  1. 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).

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