Difference between revisions of "Team:Lambert GA/Updated Part"

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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Notebook">Notebook</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Notebook">Notebook</a>
 
       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/InterLab">InterLab</a>
 
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Model">Model</a>
 
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Results">Results</a><a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Demonstrate">Demonstrate</a>
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<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Improve">Improve</a>
 
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Attributions">Attributions</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Attributions">Attributions</a>
 
        
 
        
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       <a href="https://2017.igem.org/Team:Lambert_GA/Parts" class="dropbtn">Parts</a>
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       <a class="drplink" style="transition: color 0.5s ease-in-out;"  
 
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href="https://2017.igem.org/Team:Lambert_GA/Updated_Part">Updated Parts</a>
 
href="https://2017.igem.org/Team:Lambert_GA/Updated_Part">Updated Parts</a>
 
       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Composite Part">Composite Parts</a>
 
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<center><font color= "#D49AE6"> BBa_K1911002: ClpP </font>
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<center><a href="http://parts.igem.org/wiki/index.php/Part:BBa_K1911001" style="color:#D49AE6;"> BBa_K1911001: pLac ClpXP CI </a>
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<img src="https://static.igem.org/mediawiki/2017/2/2d/R0011clpxpcidiagram.png">
 
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<i style="font-size:14px;"> pLac ClpXP CI design </i></center>
 
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The ClpP gene codes for the ClpP subunit protein which works together with the ClpX subunit to break down proteins that are tagged with degradation tags. The ClpXP system identifies tagged proteins and proceeds to unfold the protein and break it down into individual amino acids. The ClpXP system along with degradation tags can be used to break down certain proteins which are produced by certain genes of interest and it has a range of applications such as measuring how fast a certain protein can be degraded in a cell. The ClpP sequence which was pulled from the <i>E. coli</i> genome contained illegal restriction enzyme sites. In order to remove these illegal sites, wobbles were introduced into the ClpP sequence so that the gene is not cut anywhere and is allowed to function properly. ClpP is a naturally present gene in the <i>E. coli</i> genome.
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This year, Lambert iGEM improved upon the characterization of pLac ClpXP CI (part number BBa_K1911001), a part submitted by Lambert’s 2016 iGEM team. pLac ClpXP CI is the third part of their inducible construct and codes for ClpXP, a non-lysosomal protein degradation system, and CI, a competitive inhibitor that, once expressed, inhibits further transcription of p-lambda-rLacI tsPurple. Last year, there was an error in the submitted sequence where the beginning of the construct was omitted from the parts registry page. This year’s team recognized the problem and fully sequenced pLac ClpXP CI to determine the correct sequence. They began by sequencing with VF2 and VR, however since pLac ClpXP CI is almost 3000 base pairs long, the initial sequencing did not capture the middle part of the sequence. To fix this problem, primers were designed to capture the middle thousand base pairs; the results of this sequencing confirmed the middle part of the construct. After comparing this year’s sequencing results to last year’s submitted part, the team realized there was a double terminator at the beginning of the sequence, which would stop transcription from pλR LacI tsPurple, the earlier part of the construct. In light of this information, the parts registry page from last year was updated for clarity on the full construct design.
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<center><font color= "#D49AE6">BBa_K1911003: ClpX </font>
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<br><img src="https://static.igem.org/mediawiki/2017/f/f8/T--Lambert_GA--clpx.png"><br></center>
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ClpX is part of the protein-degradation system called ClpXP. It works in unison with another protein called ClpXP so that it can de-linearize and unfold proteins into amino acids which can then be recycled by the cells. In <i>E. coli</i> cells, ClpXP focuses on degrading incorrect proteins and reducing them down into amino acids which can be recycled by the cells. ClpXP recognizes the incorrect proteins by certain SsrA-tags which are attached at the ends of incorrect proteins. ClpX in unison with ClpP can be used to degrade unwanted proteins and can be used in future projects in conjunction with certain degradation tags. There were illegal restriction sites which cut the ClpX gene into smaller sequences, rendering it obsolete. So we had to introduce wobbles to eliminate these illegal restriction sites so that ClpX may be expressed and allowed to function as a complete protein. ClpX is found naturally in the <i>E. coli</i> genome.
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<center><font color= "#D49AE6">BBa_K1911005: eGFP </font>
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<br><img src="https://static.igem.org/mediawiki/2017/9/9b/T--Lambert_GA--egfp.png"><br></center>
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eGFP = Enhanced green fluorescent protein which can be used as a reporter gene. eGFP was used in our project to show the effects of attaching the DAS and LAA degradation tag to a target gene which is then degraded by the ClpXP system. In essence, the eGFP should be less prominent in <i>E. coli</i> cells which contain DAS tags and LAA tags compared to the <i>E. coli cells</i> that have constructs which do not have any degradation tags attached to the eGFP. eGFP can be used as a reporter gene and helps the observer easily see the coloration of <i>E. coli</i> cells which indicate a successful transformation. The eGFP could not contain any illegal restriction enzyme sites as it would cut the sequence up, not allowing for the expression of the eGFP protein. GFP is originally found in jellyfish <i>Aequorea victoria</i> but eGFP has been modified to be more sensitive. It helps the observers to easily determine the green coloration of the GFP protein.
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Latest revision as of 03:52, 2 November 2017


Updated Parts



BBa_K1911001: pLac ClpXP CI

pLac ClpXP CI design

This year, Lambert iGEM improved upon the characterization of pLac ClpXP CI (part number BBa_K1911001), a part submitted by Lambert’s 2016 iGEM team. pLac ClpXP CI is the third part of their inducible construct and codes for ClpXP, a non-lysosomal protein degradation system, and CI, a competitive inhibitor that, once expressed, inhibits further transcription of p-lambda-rLacI tsPurple. Last year, there was an error in the submitted sequence where the beginning of the construct was omitted from the parts registry page. This year’s team recognized the problem and fully sequenced pLac ClpXP CI to determine the correct sequence. They began by sequencing with VF2 and VR, however since pLac ClpXP CI is almost 3000 base pairs long, the initial sequencing did not capture the middle part of the sequence. To fix this problem, primers were designed to capture the middle thousand base pairs; the results of this sequencing confirmed the middle part of the construct. After comparing this year’s sequencing results to last year’s submitted part, the team realized there was a double terminator at the beginning of the sequence, which would stop transcription from pλR LacI tsPurple, the earlier part of the construct. In light of this information, the parts registry page from last year was updated for clarity on the full construct design.