Team:UFlorida/Improve
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Improve
Part Improvement: BBa_K1587004
We attempted to improve Toulouse 2015’s Butyrate synthesis pathway, BBa_K1587004. A critical analysis of this pathway reveals that the ccr gene, which catalyzes the reduction of Crotonyl-coA to Butyryl-CoA, is NADPH-dependent. Without an enzyme that couples NADH to NADPH, this pathway will not be functional for butyrate production. Our rationale for gene deletions was to force E. Coli to produce butyrate with our synthetic pathway in order to maintain redox balance, but this would be impossible without an enzyme that catalyzes the reduction using NADH instead of NADPH. For this reason, we attempted to add the Trans-enoyl reductase (Ter) to the pathway via restriction digestion to form a complete redox balanced pathway that could be driven further via gene deletions.
The process for the assembly required us to utilize our failed gblock that was synthesized for the original attempts at creating a functional pathway in E. coli Nissle. We cloned the Ter CDS out of plasmid sequence donated by one of our team mentors, Dr. Shanmugan. The biobrick BBa_I0500 containing pBAD/araC promoter was taken from the iGEM distribution kit.