Team:Evry/Data

From 2012.igem.org

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<h3>The auxin emission system</h3>
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<h3>Auxin production device</h3>
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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.
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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>
<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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<h3>The auxin receiver system</h3>
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<h3>Auxin reception device</h3>
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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. 
<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>
<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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<h2>Reporters and promotors</h2>
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<h2>Eukaryotic construction tools and characterization systems</h2>
<h3>Summary</h3>
<h3>Summary</h3>
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<p>In our Project we planned to screen a series of different reporters  along with different tissue specific promotors. This was done to validate cross-talk between the emitter-receiver system and and screen  various reporters to elucidate the efficiency of different reporters in Tadpole manipulation.</p>
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<p>
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As well as our plasmids and hormonal system, we submitted a number of basic eukaryotic parts to the registry to help future teams develop synthetic biology in Xenopus. We also submitted several characterization constructions for other teams to test our devices.
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Revision as of 18:15, 26 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 3 new plasmid backbones for creating multicellular synthetic systems. They contain a CMV promoter: (ubiquitous expression), an elastase promoter: (pancreas specific), and a Heat-shock promoter: (Heat or stress inducible). All 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.

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

Eukaryotic construction tools and characterization systems

Summary

As well as our plasmids and hormonal system, we submitted a number of basic eukaryotic parts to the registry to help future teams develop synthetic biology in Xenopus. We also submitted several characterization constructions for other teams to test our devices.

  • K812030:Citrine reporter with a Kozak sequence for eukaryotic expression
  • K812031:sfGFP with kozak sequence for eukaryotic expression
  • K812032:mCFP with kozak sequence for eukaryotic expression
  • K812200:Biobricked pSC2+ plasmid with Elastase promoter for frogs and chicken
  • K812300:Biobricked pSC2+ plasmid with HSP70 promoter for frogs and chicken
  • K812233:sfGFP in pCS2+ downstream to elastase promoter
  • K812331:sfGFP in pCS2+ downstream to HSP70 promoter

Characterization


The information on characterized part can be found here: FrenchFrog

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

say we are testing them





















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