Virginia Tech/Updates/Plasmid/Design
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Designing the Reporter Plasmid:VT iGEM Project 2007: Engineering an Epidemic |
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UNDER CONSTRUCTION!! The Theory behind our Reporter PlasmidWe 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 DesignThe 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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