Part:BBa_K1975007:Design
PhaB fused to Red Fluorescent Protein
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1177
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 697
Illegal AgeI site found at 1421 - 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 PhaB and RFP should be directly attached or if they should have some kind of linker. After looking through different studies that have been working with PhaB 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. This linker contain a restriction site so it will be possible to digest the construct and then get rid off the fluorescent tag if it is not needed anymore.
Source
The genetic sequence for PhaB is original from Ralstonia eutropha genomic DNA, but has been optimized to the expression of B. subtilis. The sequence for RFP genomic DNA from Discosoma sp. This sequence has also been optimized for expression in B. subtilis.