Part:BBa_K4895004:Design
Complete Aerobic Beta-Oxidative enzymes for Unsaturated Fatty Acids in Salmonella Enterica LT2 Strai
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 3484
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Many of our considerations dealt with the length of and the possible toxicity of the enzyme to bacteria. It is found that fadA comes from a family of Beta-oxidative genes under the fadR regulon. The fadR regulon works as an apoprotein double repressor for both the fad family and fab family.
The fab family consists of other metabolically active enzymes, that instead oxidate unsaturated long chain fatty acids.
In the presence of acyl-coA's, the fadR apoprotein will unbind from fad family genes and bind to the UTR of fab family genes. However, without acyl-coA, fadR always binds to the UTR of fad family, and does not bind to fab family. As such, it is important to consider that the heavy regulation of native fad genes may become a hinderance when beta oxidation needs to be constitutively expressed. This sequence only contains the coding region, and avoids the regulation by fadR.
In terms of the genomic design, we decided to utilize native e.coli UTR regions. The first is taken from genomic fadba, to best emulate the expression and coupling of that in e.coli. The second UTR is taken from Lacz/y, which is generally recognized as a consistent UTR with a good RBS. Furthermore, this composite part was constructed from three different ~2kb inserts, with primers ligating the inserts within the UTR's, thus avoiding messy recombination/ligation.
Source
Genomic sequence of Salmonella Enterica LT2 strain. Member of the fad family of beta-oxidative enzymes. This sequence is adapted from amino acid sequences before being codon-optimized to fit the transcriptional needs of E.Coli. The codon optimization was performed through Benchling.