Virginia Tech/plasmid design

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'''<html><font color="#660033" size="6"><center>Designing the Reporter Plasmid</center></font></html>'''
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<h1>Designing the Reporter Plasmid</h1>
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[[Image:Gene_circuit.PNG|thumb|300px|right|The right promoter of the λ switch, which we used to build our plasmid. We replaced the cI and Cro genes with fluorescent protein genes on our plasmid.]]
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<p><html><h3>The Theory behind our Reporter Plasmid</h3></html>
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<h3>For our models, we needed to know how the virus infects a cell prior to seeing symptoms.</h3>
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We need to know how the virus infects a cell prior to seeing symptoms. We decided to utilize the proteins that the virus naturally makes upon infection: cI and Cro. The decision to go lysogenic or lytic is determined by a number of other proteins and conditions, but these are the final two proteins involved in the decision. Thus, for our reporter plasmid, we decided to use one of the promoters from bacteriophage lambda. This bistable promoter makes a decision based on the presence of these two proteins. However, on the reporter plasmid, instead of promoting the lytic or lysogenic pathway, the promoter will promote a different fluorescent gene depending on which protein predominated. </p>
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<p><html><h3>Challenges in Design</h3></html>
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We decided to utilize the proteins that the virus naturally makes upon infection: cI and Cro. The decision between lysogeny and lysis is determined by a number of other proteins and conditions, but these are the final two proteins involved in the decision. Thus, for our reporter plasmid, we decided to use one of the promoters from bacteriophage lambda. This bistable promoter makes a decision based on the presence of these two proteins. However, on the reporter plasmid, instead of promoting the lytic or lysogenic pathway, the promoter will promote a different fluorescent gene depending on which protein is predominating.
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The lambda switch has two main regions: a left side and a right one. Each region contains two promoters that promote in opposite directions. We know that cI and Cro bind to the operators of the right region but they will also form a complex with the left one. We needed to use the promoters on the right region, but we were not sure to what extent binding on the left would affect our promoters. Thus, we decided to leave off the left region assuming that the promoters on the right will work just fine without it. </p>
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<p>One of the two promoters in our right region, Prm, did not have a ribosome binding site. Therefore, translation started right after the +1 site. The other promoter, Pr, did have a ribosome binding site. The discrepancy between ribosome binding sites is the primary reason that we designed so many constructs; we weren't sure which would work. Another reason for the extra constructs was to compare the different approaches to synthetic biology: the iGEM approach of ligating with a prefix and suffix, and a more traditional approach with other restriction sites. </p>
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'''Challenges in Design'''
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The lambda switch has two main regions: a left side and a right one. Each region contains two promoters that promote in opposite directions. We know that cI and Cro bind to the operators of the right region but they will also form a complex with the left one. We needed to use the promoters on the right region, but we were not sure to what extent binding on the left would affect our promoters. Thus, we decided to leave off the left region assuming that the promoters on the right will work just fine without it.
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<p><html><h3>The Four Promoter Constructs</h3></html></p>
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One of the two promoters in our right region, Prm, did not have a ribosome binding site. Therefore, translation started right after the +1 site. The other promoter, Pr, did have a ribosome binding site. To account for our uncertainty about RBS, we designed a multiple different promoters.
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<p>KEY:</p>
 
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<p>Turquise: -35 sites<br>
 
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Bright Green: -10 sites<br>
 
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Pink: +1 sites<br>
 
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Dark Green: Translational STOP codon frequently found in E. coli.<br>
 
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Yellow: Shine Dalgarno Sequence (Beginning of Ribosome Binding Site - RBS)<br>
 
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Colored/Underlined & Italisized: Restriction sites<br>
 
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Bold & Underlined: Operators</p>
 
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<p><br><br>'''Construct 1: pSBVT0001'''</p>
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<h3>KEY:</h3>
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'''Turquise:''' -35 sites<br>
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'''Bright Green:''' -10 sites<br>
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'''Pink:''' +1 sites<br>
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'''Dark Green:''' Translational STOP codon frequently found in E. coli.<br>
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'''Yellow:''' Shine Dalgarno Sequence (Beginning of Ribosome Binding Site - RBS)<br>
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'''Colored/Underlined & Italisized:''' Restriction sites<br>
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'''Bold & Underlined:''' Operators
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'''Construct 1: pSBVT0001'''
[[Image:PSBVT0001.JPG | 500px]]
[[Image:PSBVT0001.JPG | 500px]]
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<p><br><br>'''Construct 2: pSBVT0002'''</p>
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'''Construct 2: pSBVT0002'''
[[Image:PSBVT0002.JPG | 500px]]
[[Image:PSBVT0002.JPG | 500px]]
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<p><br><br>'''Construct 3: pSBVT0003'''</p>
 
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[[Image:PSBVT0003.JPG | 500px]]
 
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<li>Restriction Sites: BspHI and NcoI - from left to right respectively. The restriction sites were designed around two commercial reporter genes that we had on hand: AcGFP and DsRed. Both have NcoI sites right at the start of the sequence for the gene. The BspHI is compatible with NcoI and it allows for easy screening once the plasmid is constructed.</li>
 
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<li>A new RBS, which was modified from the original Pr RBS so that it would work with the two new restriction sites.</li>
 
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<li>Translational stop codon six base pairs after the +1 site on the Prm side.</li>
 
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<p><br><br>'''Construct 4: pSBVT0004'''</p>
 
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[[Image:PSBVT0004.JPG | 500px]]
 
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<li>Restriction Sites: BspHI and NcoI - from left to right respectively.</li>
 
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<li>The RBS from pSBVT0003 has been modified to be A-T rich, versus the previous RBS which has a number of G-C base pairs. This should insure better expression of our reporter genes. </li>
 
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<li>Translational stop codon six base pairs after the +1 site on the Prm side.</li>
 
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Latest revision as of 03:23, 27 October 2007

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Designing the Reporter Plasmid

The right promoter of the λ switch, which we used to build our plasmid. We replaced the cI and Cro genes with fluorescent protein genes on our plasmid.

For our models, we needed to know how the virus infects a cell prior to seeing symptoms.

We decided to utilize the proteins that the virus naturally makes upon infection: cI and Cro. The decision between lysogeny and lysis is determined by a number of other proteins and conditions, but these are the final two proteins involved in the decision. Thus, for our reporter plasmid, we decided to use one of the promoters from bacteriophage lambda. This bistable promoter makes a decision based on the presence of these two proteins. However, on the reporter plasmid, instead of promoting the lytic or lysogenic pathway, the promoter will promote a different fluorescent gene depending on which protein is predominating.

Challenges in Design The lambda switch has two main regions: a left side and a right one. Each region contains two promoters that promote in opposite directions. We know that cI and Cro bind to the operators of the right region but they will also form a complex with the left one. We needed to use the promoters on the right region, but we were not sure to what extent binding on the left would affect our promoters. Thus, we decided to leave off the left region assuming that the promoters on the right will work just fine without it.

One of the two promoters in our right region, Prm, did not have a ribosome binding site. Therefore, translation started right after the +1 site. The other promoter, Pr, did have a ribosome binding site. To account for our uncertainty about RBS, we designed a multiple different promoters.


KEY:

Turquise: -35 sites
Bright Green: -10 sites
Pink: +1 sites
Dark Green: Translational STOP codon frequently found in E. coli.
Yellow: Shine Dalgarno Sequence (Beginning of Ribosome Binding Site - RBS)
Colored/Underlined & Italisized: Restriction sites
Bold & Underlined: Operators


Construct 1: pSBVT0001

PSBVT0001.JPG

  • iGEM prefix (EcoRI, NotI, XbaI) and suffix (SpeI, NotI, PstI)
  • Original RBS from Pr, which we also copied over to the Prm in case an RBS was necessary to express the fluorescent proteins.
  • Translational stop codon six base pairs after the +1 site on the Prm side so that translation will not start at the +1 like it would in the lambda genome naturally.


Construct 2: pSBVT0002 PSBVT0002.JPG

  • iGEM prefix (EcoRI, NotI, XbaI) and suffix (SpeI, NotI, PstI)
  • No extra RBS.iGEM provides ribosome binding sites as a part of its DNA registry and most of the reporter genes already have RBS built into the parts.
  • Translational stop codon six base pairs after the +1 site on the Prm side.




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