Difference between revisions of "Team:Lambert GA/Design"

 
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       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Applied_Design">Applied Design</a>
 
       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Applied_Design">Applied Design</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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The full genetic construct is comprised of three parts: p-lambda-r LacI (a CI repressible promoter), one of the three tsPurple parts (tsPurple with no deg tag, tsPurpleDAS, or tsPurpleLAA), and pLac-ClpXP-CI (a LacI repressible promoter, a proteolysis mechanism, and CI to regulate p-lambda-r).  
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The full genetic construct is comprised of three parts: pλR LacI, one of the three tsPurple parts (tsPurple with no deg tag, tsPurpleDAS, or tsPurpleLAA), and pLac ClpXP CI (a LacI repressible promoter, a proteolysis mechanism, and CI to regulate pλR).  
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Without IPTG induction, pλR is naturally expressing and will promote the LacI gene and the tsPurple. The LacI gene produces lac inhibitor proteins that bind to the pLac1 site and inhibit pLac from promoting ClpXP and the CI. This system allows tsPurple to be significantly expressed without any degradation in the <i>E. coli</i> cells. Upon induction of IPTG, IPTG molecules will bind to the LacI repressor, which prevents the lac inhibitor proteins from repressing pLac and allows for expression of ClpXP and the CI. Once expressed, the CI will repress the pλR promoter to ensure that no additional lac inhibitor proteins (or tsPurple chromoproteins) are being produced. Simultaneously, ClpXP will proceed to degrade the chromoprotein tsPurple when a degradation tag (in our system, either DAS or LAA) is attached.
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Ideally, the final result would be three fully assembled constructs: pλR LacI tsPurple pLac ClpXP CI, pλR LacI tsPurpleDAS pLac ClpXP CI, and pλR LacI tsPurpleLAA pLac ClpXP CI. Once all three constructs were induced with IPTG, the relative levels of degradation would be compared; theoretically the construct with no degradation tag would show no degradation, the DAS tag (which is a moderately fast degradation tag) would show a middle level of degradation, and the LAA tag (a fast degradation tag) would show the highest levels of degradation. The Chrome-Q system would then be utilized to measure the HSV values of the three different levels of degradation.
  
 
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Without IPTG induction, p-lambda-r is naturally expressing and will promote the LacI gene and the tsPurple. The LacI gene produces lac inhibitor proteins that bind to the pLac1 site and inhibit pLac from promoting ClpXP and the CI. This system allows tsPurple to be significantly expressed without any degradation in the <i>E. coli</i> cells. Upon induction of IPTG, IPTG molecules will bind to the LacI repressor, which prevents the lac inhibitor proteins from repressing pLac and allows for expression of ClpXP and the CI. Once expressed, the CI will repress the p-lambda-r promoter to ensure that no additional lac inhibitor proteins (or tsPurple chromoproteins) are being produced. Simultaneously, ClpXP will proceed to degrade the chromoprotein tsPurple when a degradation tag (in our system, either DAS or LAA) is attached.
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Ideally, the final result would be three fully assembled constructs: p-lambda-r-LacI-tsPurple-pLac-ClpXP-CI, p-lambda-r-LacI-tsPurpleDAS-pLac-ClpXP-CI, and p-lambda-r-LacI-tsPurpleLAA-pLac-ClpXP-CI. Once all three constructs were induced with IPTG, the relative levels of degradation would be compared; theoretically the construct with no degradation tag would show no degradation, the DAS tag (which is a moderately fast degradation tag) would show a middle level of degradation, and the LAA tag (a fast degradation tag) would show the highest levels of degradation. The Chrome-Q system would then be utilized to measure the HSV values of the three different levels of degradation.
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With this in mind, we assembled six constructs - three with the GFP chromoprotein and three with tsPurple. The three GFP constructs served as our baseline constructs as we can compare the success of failure of the tsPurple constructs; the built  GFP constructs work to ensure that our circuit can feasibly deliver results. In each set - the separate GFP and tsPurple constructs- the first construct serves as our control due to the lack of degradation tags on the reporters. In the second construct, the reporters are associated with the DAS degron; and in the third construct, the reporters were tagged with the LAA degron. According to the biobrick registry, LAA,  is a much stronger degradation tag than DAS; consequently, we expect to see a greater degree of degradation (significantly lighter color of purple) in the constructs with the LAA tag than the constructs with the DAS tag and no degradation tags.
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<h2>Construct Design</h2>
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    <iframe id="pdf-viewer" src="https://static.igem.org/mediawiki/2017/9/93/Constructdesignslidespdf.pdf" frameborder="0" style="height:1000px;width:600px;"> Cloning Workflow</iframe>
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Moreover, we have assembled constructs in two different knockout strains:  
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<LI style= "color: white; font-size: 20px;"><u>Keio ClpX Knockout:</u> In this strain, ClpX will be knocked out and ClpP will be allowed to function. ClpX is responsible for de-linearizing the target protein into its primary structure and then translocating it into a proteolytic cavity in ClpP. Since ClpX will not be expressed, the chromoprotein will not be de-linearized and hence prevent ClpP from degrading the primary structure of the protein. With this in mind, we expect our plates to remain purple in the Keio ClpX Knockout strain.
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<LI style= "color: white; font-size: 20px;"><u>Keio ClpP Knockout:</u> In this strain, ClpP will be knocked out and ClpX will be allowed to function. ClpP is responsible for degrading the primary structure of the protein itself; it does this by breaking the individual covalent bonds (polypeptide bonds) that exist between the amino acids in the polypeptide chain. Since ClpP will not be expressed, the chromoprotein will not be degraded into individual amino acids. However, despite this, the chromoprotein will still be degraded partially because it will be de-linearized by ClpX. In other words, although the protein will still be in its primary structure, it will still lack the hydrogen bonds, hydrophobic interactions, ionic bonds, disulfide bridges, and R-Group interactions that exist in its tertiary (or in some cases, quaternary) structure; consequently, that will limit the chromoprotein’s ability to express its pigments. With this in mind, we expect our plates to have a smaller number of purple cells present in the Keio ClpP Knockout strain.
 
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Latest revision as of 16:09, 16 December 2017


Design


The full genetic construct is comprised of three parts: pλR LacI, one of the three tsPurple parts (tsPurple with no deg tag, tsPurpleDAS, or tsPurpleLAA), and pLac ClpXP CI (a LacI repressible promoter, a proteolysis mechanism, and CI to regulate pλR).






Without IPTG induction, pλR is naturally expressing and will promote the LacI gene and the tsPurple. The LacI gene produces lac inhibitor proteins that bind to the pLac1 site and inhibit pLac from promoting ClpXP and the CI. This system allows tsPurple to be significantly expressed without any degradation in the E. coli cells. Upon induction of IPTG, IPTG molecules will bind to the LacI repressor, which prevents the lac inhibitor proteins from repressing pLac and allows for expression of ClpXP and the CI. Once expressed, the CI will repress the pλR promoter to ensure that no additional lac inhibitor proteins (or tsPurple chromoproteins) are being produced. Simultaneously, ClpXP will proceed to degrade the chromoprotein tsPurple when a degradation tag (in our system, either DAS or LAA) is attached.


Ideally, the final result would be three fully assembled constructs: pλR LacI tsPurple pLac ClpXP CI, pλR LacI tsPurpleDAS pLac ClpXP CI, and pλR LacI tsPurpleLAA pLac ClpXP CI. Once all three constructs were induced with IPTG, the relative levels of degradation would be compared; theoretically the construct with no degradation tag would show no degradation, the DAS tag (which is a moderately fast degradation tag) would show a middle level of degradation, and the LAA tag (a fast degradation tag) would show the highest levels of degradation. The Chrome-Q system would then be utilized to measure the HSV values of the three different levels of degradation.



Construct Design