Part:BBa_K1975004:Design
Beta-Ketothiolase fused to Cyan Fluorescent Protein
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1882
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
To secure the optimal expression we choose to make a codon optimization of the genes. This was done through the Codon Optimization tool at IDT's webpage. Besides that we looked through the literature to figure our if the PhaA and CFP should be directly attached or if they should have some kind of linker. After looking through different studies that have been working with PhaA in Escherichia coli we came to the conclusion that a linker should not be necessary to secure high expression and activity. We therefore decided to only make a small linker (6 bp) between the gene and the tag.
Source
The genetic sequence for PhaA is original from Ralstonia eutropha genomic DNA, but has been optmiized to the expression of B. subtilis. The sequence for CFP genomic DNA from Acropora aculeus. This sequence has also been optimized for expression in B. subtilis.
References
Taguchi et. al. 2008: "A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme", PNAS