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Team:Calgary/Sandbox
From 2010.igem.org
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Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein. Our system detects possible errors in these three steps using two circuits. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.
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