Virginia Tech/Updates/Plasmid/Design

From 2007.igem.org

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<center><h1><font color="#ff8c00"> Reporter Plasmid Updates:  
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<center><h1><font color="#ff8c00"> Designing the Reporter Plasmid:
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<center><h3><font color="#8b0000"> VT iGEM Project 2007: Engineering an Epidemic</font></h3></center>
<center><h3><font color="#8b0000"> VT iGEM Project 2007: Engineering an Epidemic</font></h3></center>
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<p><h3>Introduction: The Reporter Plasmid</h3>
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<p>UNDER CONSTRUCTION!!</p>
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In order to collect data for our experiments, we needed a way to tell whether an infected cell was lysogenic or lytic. We decided that a good way to do this would be to use a reporter plasmid containing the lambda promoter and two fluorescent genes. The same chemicals that decided which pathway the cell entered would decide which fluorescent protein from our reporter plasmid to express.</p>
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[[Image:Lambda_Pr_rep.JPG]]
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<p><h3>The Theory behind our Reporter Plasmid</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><h3>Updates</h3></p>
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<p><h3>Challenges in Design</h3>
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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>'''Designing the Promoter Construct'''<br>
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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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''July 24, 2007''</p>
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<p>Click [[ here]] to read about how we designed the reporter plasmid's main feature, the promoter construct.</p>
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<p><h3>The Four Promoter Constructs</h3></p>
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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><h3>Construct 1:</h3></p>
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[[Image:]]
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This construct contains the iGEM prefix (EcoRI, NotI, XbaI) and suffix (SpeI, NotI, PstI). It also contains the original RBS from Pr, which we also copied over to the Prm. We added a ribosome binding site to the Prm in case a RBS was necessary to express the fluorescent proteins.  We also added the translational stop codon six basepairs after the +1 site so that translation would not start at the +1 like it would in the lambda genome naturally.
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This promoter is an exact replicate of the first promoter, but without the ribosome binding sites. The reason for this is because 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.
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This promoter takes a more traditional approach to synthetic biology. It contains the STOP codon that all of the previous promoters contain as well as a new RBS. This RBS was modified from the original Pr RBS so that it would work with the two new restriction sites (BspHI and NcoI - from left to right respectively). The restriction sites were designed around the two commecial 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.
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This promoter is exactly the same as the third promoter, but for one difference. The RBS has been modified to be AT rich, verces the previous RBS which has a number of GC basepairs. This should insure better expression of our reporter genes.
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Revision as of 17:40, 24 July 2007

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Designing the Reporter Plasmid:

VT iGEM Project 2007: Engineering an Epidemic

UNDER CONSTRUCTION!!

The Theory behind our Reporter Plasmid

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.

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. 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.


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