Team:Valencia/prototypes

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Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br>
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br>
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<center><img src="https://2012.igem.org/File:VLC_transformingcoccus.JPG"></center>
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<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center>
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<center><b>Figure 1.</b> Our idea of the construct</center>
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Parts Information:<br><br>
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<b>Parts Information</b>:<br><br>
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BBa_K754000 (link): <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br>
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<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br>
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BBa_Q04510 (link): Consists in the next parts:<br>
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<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br>
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- B0034: RBS <br>
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- C0051: cI repressor from E. coli phage lambda <br>
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<ul style="list-style-type: square">
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Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br>
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<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li>
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- B0015: double terminator<br>
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</ul>
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This is the most commonly used terminator. It seems to be reliable<br>
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-R0051: promoter (lambda cI regulated)<br>
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<ul style="list-style-type: square">
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The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br>
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<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda  
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BBa_C0061 (link): Autoinducer synthetase for AHL<br>
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Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li>
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</ul>
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<ul style="list-style-type: square">
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<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br>
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This is the most commonly used terminator. It seems to be reliable<br></li>
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</ul>
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<ul style="list-style-type: square">
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<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br>
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The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li>
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</ul>
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<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br>
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br>
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br>
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BBa_B1002 (link): Terminator<br>
 
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- Artifical terminator with %T≈85<br>
 
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- 6bp stem, 4nt loop<br>
 
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<strong>Constructing the BioBrick</strong>
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<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br>
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We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo).
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We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.  Read the protocol we followed here.
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<br><br>
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<strong>Constructing the BioBrick</strong>
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<br><br>
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We've ligated the part <a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> (psbAI promoter) with the psb1C3 vector.
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<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center>
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<center><b>Figure 2.</b> psbAI promoter into the psb1C3 vector</center>
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Latest revision as of 02:29, 27 September 2012



Designed Biobricks


Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the psbAI promoter which binds to the lambda promoter inhibiting the luxI transcription, when psbAI is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.

Figure 1. Our idea of the construct
Parts Information:

BBa_K754000 : S. elongatus PCC7942 psbAI promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.
BBa_Q04510 : Consists in the next parts:
  • C0051: cI repressor from E. coli phage lambda Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.
  • B0015: double terminator
    This is the most commonly used terminator. It seems to be reliable
  • R0051: promoter (lambda cI regulated)
    The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.
BBa_C0061 : Autoinducer synthetase for AHL
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
BBa_B1002: Terminator


Constructing the BioBrick

We've ligated the part BBa_K754000 (psbAI promoter) with the psb1C3 vector.
Figure 2. psbAI promoter into the psb1C3 vector

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