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>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Hardware">Hardware</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Hardware">Hardware</a>
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      <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Measurement">Measurement</a>
 
 
       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Model">Model</a>
 
       <a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Model">Model</a>
 
<a class="drplink" style="transition: color 0.5s ease-in-out;" href="https://2017.igem.org/Team:Lambert_GA/Software">Software</a>
 
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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 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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<img style="width:600px align:center" src="https://static.igem.org/mediawiki/2017/2/21/Clip%27dcharacterizingclpxp.PNG">
 
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<i style="font-size: 14px; color: white;">The animation shows the function of the full construct in steps:
 
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1. p-lambda-r expresses LacI, which produces lac inhibitor proteins to repress pLac, and tsPurple chromoproteins
 
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2. IPTG is introduced into the cell and binds with the lac inhibitor proteins, preventing repression of pLac and beginning expression of ClpXP and CI
 
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3. CI prevents any further expression of LacI and tsPurple, while ClpXP recognizes the degradation tag and breaks down tsPurple.
 
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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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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: 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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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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<h2>Keio Competent Cells</h2>
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<h2>Construct Design</h2>
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<u>Keio Knockout Strains:</u> To prove our genetic construct, Lambert iGEM successfully made competent cells of Keio Wild and Keio ClpP Knock-Out E. Coli strains from a dehydrated disk donated to the team by the E. Coli Genetic Stock Center. These strains naturally have the ClpXP gene (which is a two part mechanism that degrades proteins with degradation tags attached). ClpX recognizes the degradation tag and linearizes the protein, then ClpP breaks it down into individual amino acids. Theoretically, once the full genetic construct is inserted in the cell, the Keio Wild strain would have low levels of expression of the Ts-Purple due to ClpXP degradation. The Keio ClpP knock-out is expected to produce a moderate level of expression, since ClpX would linearize the protein but ClpP is not able to finish the degradation process. In contrast, Keio ClpX Knock-Out would have the most color expression since ClpX is not present to linearize the tagged protein. Thus, ClpP is not able to separate the protein into amino acids, and no degradation would occur. Unfortunately, due to time constraints Lambert iGEM was only able to insert part of the genetic construct, a promoter and Ts-Purple, into the knock-out strains and have not yet achieved the expected results.
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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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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