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Team:Yale/Project
From 2013.igem.org
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2013 Project Overview
We aim to develop an orthogonal translation system for selective incorporation of selenocysteine, a non-standard amino acid, in E. coli and use the system to produce a more effective heavy metal biosorbent.
Project Background
In 2011, Dr. Farren Isaacs and others reported progress on replacing all TAG stop codons with TAA in Escherichia coli, enabling reassignment of the TAG codon. (Isaacs et al. 2011). Dr. Isaacs has recently completed this work, creating the first genomically-recoded organism with an unassigned codon (Lajoie, M. J et al. 2013. Science. In press.).
This genome-wide recoding was enabled by lambda-Red recombineering (Sharan et al. 2009) and multiplex automated genome engineering (Wang et al. 2009). λ-Red recombineering uses the lambda prophage protein Beta to mediate the homologous recombination of an exogenous single-stranded oligonucleotide, delivered into the cell by electroporation, with the host genome, by annealing that oligonucleotide to single-stranded genomic DNA exposed on the lagging strand of the replication fork (Ellis et al. 2001). A 30-bp region of homology at either end of a sequence is sufficient to drive recombination of the entire strand, including mismatched regions, and mismatches thus incorporated are passed to progeny at high efficiency (up to 30%) in strains lacking methyl-directed mismatch repair (ΔmutS; Constantino & Court 2003).
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