Team:Evry/Data

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'''Auxin Degron System in Iadpoles'''
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<h1>Data page: Summary of our summer work</h1>
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Our System is based on a ligand-receptor communication where auxin is produced in a two step manner. following production, when auxin interacts with the auxin-inducible degradation(AID), the bound protein to the '''AID''' is thereafter degraded. In our system the AID-bound protein is the famous GFP reporter which upon loss of fluorescence, indicates the system's fidelity    ).
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''The following Diagram is a schematic representation of the '''Auxin-sensing System''' '''along with the main necessary interacting parts in the system submitted by our team:
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<center><img src="https://static.igem.org/mediawiki/2012/c/cd/Frog_tube.png"></center>
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<br />
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| '''Data For Our Favorite New Parts'''
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<h2>French Froggies : New Xenopus plasmids for creating multicellular systems</h2>
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# [http://partsregistry.org/Part:BBa_K812000 Main Page] - '''Eukaryotic exoression vector for tadpole injections , BBa_K812000''': This vector has all the prerequisites for eukaryotic trasncriptiona like 5' and 3' Untranslated regions implicated in effective transcriptional regulations . On top of this, it has all the requirements for amplification of any casette in bacteria.
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# [http://partsregistry.org/Part:BBa_BBa_K812000 Main Page] - '''Auxin production cassette, BBa_K812000''': The Ligand Production Cassette
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<center><img src="https://static.igem.org/mediawiki/2012/c/cc/Plasmid_backbone.png" alt="Image unavailable" width="550px" /></center>
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<p>We developed and submitted to the registry 2 new plasmid backbones for creating multicellular synthetic systems and the corresponding biobricked promoters<p>
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<ul>
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<li>The pSC2+ with the CMV promoter: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812000">K812000</a></li>
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<li>The pSC2+ with pElastase promoter (pancreas specific): <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812200">BBa_K812200</a></li>
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<li>And the Biobricked pElastase alones: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812233">BBa_K812233</a></li>
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<li>And with a fluorescent citrine to characterize the promoter pElastase in the frog: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812331"></a></li>
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<li>We also submitted a heat shock Xenopus promoter: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812331">BBa_K812331</a></li>
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</ul>
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'''''Auxin Production System''''[[Image:DEGRON_PART_1_.jpg]]
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<h2>A serie of new eukaryotic reporters ready for expression</h2>
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[[File:Sélection_168.png|thumb|right|Expression of the sfGFP in pCS2+ microinjected in a tadpole]]
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<p>We also prepared a series of fluorescent reporters with a kozak sequence for high expression in eukaryotes.</p>
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# [http://partsregistry.org/Part:BBa_K812013 Main Page] - '''BAR Bad Odor Receptor, BBa_K812013''': The Auxin -induced degradation system of GFP which includes the GFP fused with AID upstream to OSTir1 which upon expression they form the auxin degradation system. Consequently, after interaction of auxin GFP will be degraded
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<ul>
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<li>A kozak-citrine in pSB1C3: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812030">K812030</a></li>
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<li>A kozak-sfGFP in pSB1C3: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812031">K812031</a></li>
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<li>A kozak-mCFP in pSB1C3: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812032">K812032</a></li>
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</ul>
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<p>And provided them directely cloned in pSC2+ for direct expression in Xenopus or chicken:</p>
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<ul>
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<li>The Citrine in pSC2+: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812130">K812130</a></li>
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<li>The mCFP in pSC2+: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812132">K812132</a></li>
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<li>The sfGFP in pSC2+: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812133">K812133</a></li>
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</ul>
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<p>All these plasmids can be directly injected in to the <i>Xenopus</i> embryo after a miniprep, and are designed for rapid testing of multicellular devices. They contain all sequences necessary for a tissue specific expression, as well as debugging tools. Several plasmids can be co-injected, allowing ratio adjustment and linking the system do modeling.Once the system is tested and debugged, the final system can be implemented by chromosomal insertion. </p>
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'''Auxin-Induced Degradation System Response Cassette'''<br>[[Image:Degron part2.jpg]]
 
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<h3>Characterization</h3>
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</br>
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The information on characterized part can be found here: <a href="https://2012.igem.org/Team:Evry/FrenchFrog">FrenchFrog</a>
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<h2>The auxin system</h2>
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| '''Golden Brick Parts'''
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# [http://partsregistry.org/Part:BBa_J45119:Experience Experience] - '''Wintergreen odor enzyme generator, BBa_J45119''' (MIT, iGEM 2006): 98 out of 100 volunteer subjects standing up to 5 feet away from the bacterial cultures could distinguish wintergreen-producing bacteria from negative controls.
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# [http://partsregistry.org/Part:BBa_J61110:Experience Experience] - '''RBS, BBa_J61110''' (Arkin Lab, 2007): Of the 5 RBS Parts we tested, this RBS works best for expressing yellow fluorescent protein-tagged BAR
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| [[Image:KAH_053111_textbubble3.jpg‎|Text bubble 3]]
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| '''We've Also Characterized the Following Parts'''
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# [http://partsregistry.org/Part:BBa_XXXXX Main Page] - '''Air Freshilizor, BBa_XXXXX''': Our mathematical model predicts that the threshold of activation is 10 parts per billion, the concentration of Butanethiol that humans can typically smell
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| [[Image:KAH_053111_textbubble4.jpg‎|Text bubble 4]]
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{| style="background: #A8E4A0;"
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<h3>Auxin production device</h3>
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<p>
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|In this example, this fictional team ran out of time before they could test the complete device and use it as a synthetic air freshener. However, they made progress testing their new promoter by using GFP and they did some mathematical modeling to understand the behavior of the system. They also conducted a test to see how far the wintergreen scent could travel from a flask of culture.<br><br>
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We adapted the auxin production module created by the Imperial College 2011 team for it to be used in eukaryotes. We made a system designed for coinjection, and a system which works with a single “operon” using pep2A. This is a short self-cleaving peptide, which will cut a nascent peptide chain to make 2 proteins, insuring stochiometry of the products in the cell. These auxin production devices are designed to communicate through the organism with the auxin reception device.</p>
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="/DEGRON_PART_1_.jpg‎" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/4/4b/DEGRON_PART_1_.jpg" width="500" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/DEGRON_PART_1_.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 3: Schematic representation of the Auxin two-step production cassette.</div></div></div></div>
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<p> <ul>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812020">K812020: </a>IAAH enzyme that catalyzes the second and last step towards auxin production
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812021">K812021: </a>IAAM enzyme that catalyses the transformation of Tryptophan to Inodle-3-acetamide
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812014">K812014: </a>Auxin production cassette containg both enzymes above which is monocystronic
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</ul>
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</p>
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<h3>Auxin reception device</h3>
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<p>
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The auxin reception device is composed of two components : Tir1, an auxin dependant ubiquitin ligase, and AID – a peptide that is recognized and ubiquitinated specifically by Tir1, when auxin is present. By fusing AID to any protein, this protein will be degraded along with the AID tag when auxin is added to the medium.</p>
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:502px;"><a href="/File:Degron_part2.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c4/Degron_part2.jpg" width="500" class="thumbimage" /></a>  <div class="thumbcaption"><div class="magnify"><a href="/File:Degron_part2.jpg" class="internal" title="Enlarge"><img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /></a></div>Fig 2: schemetic representation of the Auxin-receiver system</div></div></div></div>
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<p>
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<ul>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812010">K812010:</a> GFP fusion with the ubuquitinase E3 OsTirI recoginition domain which a main part of the degron system
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812012">K812012:</a> OsTirI Ubiquitinase E3 for AID tagged protein degradation in the presence of auxin
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812013">K812013:</a> The Auxin reciever system golden gate-assembled containing GFP-AID OsTirI polysistronic system for auxin detection in tadpole
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</ul>
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</p>
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<h2>Goldenbricks</h2>
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<h3>Summary</h3>
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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 4: Summary of the GoldenBrick procedure</div></div></div>
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<p>The GoldenBrick is a new assembly method for the partsregistry. If you want to know more, <a href="https://2012.igem.org/Team:Evry/GB">see this page.</a></p>
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<h3>Our favourites parts</h3>
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<ul>
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<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</li>
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<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</li>
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</ul>
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<h3>The other parts we have created</h3>
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<ul>
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<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</li>
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<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</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812053">K812053:</a> A GoldenBricked version of the strong RBS B0034</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812054">K812054:</a> A GoldenBricked version of the RFP E1010</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812055">K812055:</a> A GoldenBricked version of the terminator B0015</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812056">K812056:</a> A GoldenBricked version of the pLac R0010 promoter</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812057">K812057:</a> A GoldenBricked of an sfGFP protein</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812058:</a> A GoldenBricked of medium strenght RBS J61107</li>
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K812058">K812059:</a> A GoldenBricked of week RBS J61117</li>
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</ul>
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Latest revision as of 03:40, 27 September 2012

Data page: Summary of our summer work



French Froggies : New Xenopus plasmids for creating multicellular systems

Image unavailable

We developed and submitted to the registry 2 new plasmid backbones for creating multicellular synthetic systems and the corresponding biobricked promoters

  • The pSC2+ with the CMV promoter: K812000
  • The pSC2+ with pElastase promoter (pancreas specific): BBa_K812200
  • And the Biobricked pElastase alones: BBa_K812233
  • And with a fluorescent citrine to characterize the promoter pElastase in the frog:
  • We also submitted a heat shock Xenopus promoter: BBa_K812331

A serie of new eukaryotic reporters ready for expression

Expression of the sfGFP in pCS2+ microinjected in a tadpole

We also prepared a series of fluorescent reporters with a kozak sequence for high expression in eukaryotes.

And provided them directely cloned in pSC2+ for direct expression in Xenopus or chicken:

All these plasmids can be directly injected in to the Xenopus embryo after a miniprep, and are designed for rapid testing of multicellular devices. They contain all sequences necessary for a tissue specific expression, as well as debugging tools. Several plasmids can be co-injected, allowing ratio adjustment and linking the system do modeling.Once the system is tested and debugged, the final system can be implemented by chromosomal insertion.

Characterization


The information on characterized part can be found here: FrenchFrog

The auxin system

Auxin production device

We adapted the auxin production module created by the Imperial College 2011 team for it to be used in eukaryotes. We made a system designed for coinjection, and a system which works with a single “operon” using pep2A. This is a short self-cleaving peptide, which will cut a nascent peptide chain to make 2 proteins, insuring stochiometry of the products in the cell. These auxin production devices are designed to communicate through the organism with the auxin reception device.

Fig 3: Schematic representation of the Auxin two-step production cassette.

  • K812020: IAAH enzyme that catalyzes the second and last step towards auxin production
  • K812021: IAAM enzyme that catalyses the transformation of Tryptophan to Inodle-3-acetamide
  • K812014: Auxin production cassette containg both enzymes above which is monocystronic

Auxin reception device

The auxin reception device is composed of two components : Tir1, an auxin dependant ubiquitin ligase, and AID – a peptide that is recognized and ubiquitinated specifically by Tir1, when auxin is present. By fusing AID to any protein, this protein will be degraded along with the AID tag when auxin is added to the medium.

Fig 2: schemetic representation of the Auxin-receiver system

  • K812010: GFP fusion with the ubuquitinase E3 OsTirI recoginition domain which a main part of the degron system
  • K812012: OsTirI Ubiquitinase E3 for AID tagged protein degradation in the presence of auxin
  • K812013: The Auxin reciever system golden gate-assembled containing GFP-AID OsTirI polysistronic system for auxin detection in tadpole

Goldenbricks

Summary

Fig 4: Summary of the GoldenBrick procedure

The GoldenBrick is a new assembly method for the partsregistry. If you want to know more, see this page.

Our favourites parts

  • K812050: A GoldenBricked version of pSB1C3 with J04450 as negative cloning control
  • K812051: A GoldenBricked version of pSB1K3 with J04450 as negative cloning control

The other parts we have created

  • K812050: A GoldenBricked version of pSB1C3 with J04450 as negative cloning control
  • K812051: A GoldenBricked version of pSB1K3 with J04450 as negative cloning control
  • K812053: A GoldenBricked version of the strong RBS B0034
  • K812054: A GoldenBricked version of the RFP E1010
  • K812055: A GoldenBricked version of the terminator B0015
  • K812056: A GoldenBricked version of the pLac R0010 promoter
  • K812057: A GoldenBricked of an sfGFP protein
  • K812058: A GoldenBricked of medium strenght RBS J61107
  • K812059: A GoldenBricked of week RBS J61117





































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