Team:Evry/AIDSystem

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<center><h1>Intertissue communication: <b>An orthogonal hormonal system</b></h1></center>
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<h4><p>We adapted the auxin production device from the iGEM team Imperial college 2011 to eukaryotes and combined it with an auxin detection module. This way, we created the first synthetic hormonal system for inter-tissues communication.</p></h4>
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<p>Auxin is a molecule of choice for working on tadpole. It is amphiphilic and hydrophobic so we assume it can cross the biological membrane easily but its toxicity is reported to be very weak. It can be synthetized in a two step pathway from a tryprophane precursor.</p>
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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 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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<a name="auxin" /><h2>Auxin production devices</h2></a>
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<p>We designed three auxin production devices in embryos. The devices 1 and 2 were designed to be expressed in embryos, while device 3 was designed to be expressed in <i>E. coli</i>. 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 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 meant for the co-expression of IaaH and IaaM genes in the same cells in embryos.  
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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 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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Auxin represents a family of plant hormones that control gene expression during many aspects of growth and development.</br>
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</ul>
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Figure 1 show how this system works:</br>
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<center>
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</br>
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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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<center><img src="https://static.igem.org/mediawiki/2012/8/8d/Aid_system.jpg"/></center>
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<h3>Pathway</h3>
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<center><u>Figure 1: Schematic illustration of the AID system</u></center>
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<center>
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</br>
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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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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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<a name="AID" /><h2>Auxin reception devices</h2></a>
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The AID plasmid is composed like it shows in this figure:</br>
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<p>One key requirement for the creation of a synthetic hormonal system was to find a cytosolic hormone receptor. We came across a very ingenious system developped my Masato Kanemaki laboratory coming from rice and that has been reported to work and patented in mammalian cells but not in tadpoles.</p>
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</br>
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<p>We designed two auxin production devices in embryos. To visualize the communication between different tissues or between <i>E. coli</i> and a tissue of the embryo, 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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<center><img src="https://static.igem.org/mediawiki/2012/f/fa/Aid_plasmid.jpg"/></center>
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<ul>
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<center><u>Figure 2: Schematic composition of AID plasmid</u></center>
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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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</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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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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</ul></br>
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</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/2012/3/3b/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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</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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<h3>Auxin degron system</h3>
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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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<p>This system is based on the E3 ubiquitinase SCF-TIR1, that is capable, in the presence of auxin to recognized GFP tagged with AID degron. When auxin is present in the cytosol, TIR1 binds to the degron and recruits the E2 ubiquitinase that adds the ubiquitine tag. Then, the GFP is degraded by the proteasome, giving a decrease in the fluorescence of the tissue.</p>
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<p>To ensure this system works, we checked that <i>Xenopus tropicalis</i> possesses the <a href="http://www.xenbase.org/gene/expression.do?method=displayGenePageExpression&geneId=919630&tabId=1">Skp1</a>, <a href="http://www.xenbase.org/gene/showgene.do?geneId=945765&method=displayGeneSummary">Cul1</a> and <a href="http://www.xenbase.org/gene/showgene.do?method=display&geneId=5779766">Rbx1</a> genes.</p>
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<center>
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<img src="https://static.igem.org/mediawiki/2012/5/5a/Degron1.jpg" width="800px" alt="degron syst" />
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</center>
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<h2>Advantages</h2>
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<h2>Example: Skin-Kidney communication</h3>
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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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<p>The choice of tissues has been set on the specific properties of the tissues in term of function and blod irrigation as well as on the existance of reported functionnig tissue specific promotors. As an emitter, we chose to use the skin, and as a receiver, the kidney.</p>
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</br>
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<center>
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<center><img src="https://static.igem.org/mediawiki/2012/1/15/Comparison_degron.png"/></center>
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<img src="https://static.igem.org/mediawiki/2012/2/2d/General_hormonal_schematic.png" width="600px" alt="Skin-Kidney" />
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<center><u>Figure 4: comparison of degradation based methods to control protein expression</u></center>
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</center>
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</br>
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<p>In order to trigger the emission of the hormone in our synthetic hormonal system we wanted to dissolve a chemical in the water of the tadpole that would activate an indicible promoter. The most exposed tissue to the chemical environment is undoubtely the epithelium, because of the important surface exposed to the water. On the top of it, this tissue is highly vascularized, which is important in order to acheive a high concentration of auxin in the blood. An important library of promoter has also been identified for this tissue.</p>
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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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<p>The problem of introducing a non native hormon into the blood is that the kidney is likely to eliminate it from the blood. The kidney works as an inverted filter, in the sense that it takes out every molecule from the blood and reintroduce only the one it knows, and our hormon does not nessarily belongs to these molecule. Therefore, we can anticipate that the course of your molecule will ends up there and it will be the place where it is the most concentrated. This organ seems to be the best place for expressing our receiver system. There are also good promoters coming from the different ion channels that are know to work there.</p>
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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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</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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<h2>Conclusion</h2>
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<p>The implementation of a tissue communication underlines the interest for the use of eucaryotes such as <i>Xenopus</i> in synthetic biology. This tool could be used with any protein fused with AID signal, allowing to test it in a multitissular system.</p>
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<p>Our orthogonal hormonal system was tested in pCS2+ plasmid. Using I-Sce sites, we could insert this system onto the chromosome. This would creating a durable tissue communication system for longer term use.</p>
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<p id="references">References:</p>
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<ol>
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<li><i>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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<li>https://2011.igem.org/Team:Imperial_College_London/Tour</li>
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</ol>
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Latest revision as of 22:37, 26 October 2012

Intertissue communication: An orthogonal hormonal system

We adapted the auxin production device from the iGEM team Imperial college 2011 to eukaryotes and combined it with an auxin detection module. This way, we created the first synthetic hormonal system for inter-tissues communication.

Auxin is a molecule of choice for working on tadpole. It is amphiphilic and hydrophobic so we assume it can cross the biological membrane easily but its toxicity is reported to be very weak. It can be synthetized in a two step pathway from a tryprophane precursor.

To test this system, we co-injected plasmids expressing our production and reception devices in 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 designed three auxin production devices in embryos. The devices 1 and 2 were designed to be expressed in embryos, while device 3 was 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 embryos.
  • Auxin production device 2 : this device is composed of BBa_K812014. It is meant for the co-expression of IaaH and IaaM genes in the same cells in embryos.
  • 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

One key requirement for the creation of a synthetic hormonal system was to find a cytosolic hormone receptor. We came across a very ingenious system developped my Masato Kanemaki laboratory coming from rice and that has been reported to work and patented in mammalian cells but not in tadpoles.

We designed two auxin production devices in embryos. To visualize the communication between different tissues or between E. coli and a tissue of the embryo, 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

Auxin degron system

This system is based on the E3 ubiquitinase SCF-TIR1, that is capable, in the presence of auxin to recognized GFP tagged with AID degron. When auxin is present in the cytosol, TIR1 binds to the degron and recruits the E2 ubiquitinase that adds the ubiquitine tag. Then, the GFP is degraded by the proteasome, giving a decrease in the fluorescence of the tissue.

To ensure this system works, we checked that Xenopus tropicalis possesses the Skp1, Cul1 and Rbx1 genes.

degron syst

Example: Skin-Kidney communication

The choice of tissues has been set on the specific properties of the tissues in term of function and blod irrigation as well as on the existance of reported functionnig tissue specific promotors. As an emitter, we chose to use the skin, and as a receiver, the kidney.

Skin-Kidney

In order to trigger the emission of the hormone in our synthetic hormonal system we wanted to dissolve a chemical in the water of the tadpole that would activate an indicible promoter. The most exposed tissue to the chemical environment is undoubtely the epithelium, because of the important surface exposed to the water. On the top of it, this tissue is highly vascularized, which is important in order to acheive a high concentration of auxin in the blood. An important library of promoter has also been identified for this tissue.

The problem of introducing a non native hormon into the blood is that the kidney is likely to eliminate it from the blood. The kidney works as an inverted filter, in the sense that it takes out every molecule from the blood and reintroduce only the one it knows, and our hormon does not nessarily belongs to these molecule. Therefore, we can anticipate that the course of your molecule will ends up there and it will be the place where it is the most concentrated. This organ seems to be the best place for expressing our receiver system. There are also good promoters coming from the different ion channels that are know to work there.

Conclusion

The implementation of a tissue communication underlines the interest for the use of eucaryotes such as Xenopus in synthetic biology. This tool could be used with any protein fused with AID signal, allowing to test it in a multitissular system.

Our orthogonal hormonal system was tested in pCS2+ plasmid. Using I-Sce sites, we could insert this system onto the chromosome. This would creating a durable tissue communication system for longer term use.

References:

  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).
  2. https://2011.igem.org/Team:Imperial_College_London/Tour

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