BBa_K2657005: sYFP2 with Mutational Hotspot Removed

BBa_K2657005 is an improved version of the Biobrick BBa_K864100, which is the coding sequence for super yellow fluorescent protein (sYFP2). We increased the evolutionary stability of the sequence by removing a mutational hotspot. Originally, BBa_K864100 contained a palindromic sequence. Palindromes in the DNA cause potential hairpins, which prevent RNA polymerase from translating the DNA correctly. We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor, BBa_P10500, via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly reactions, which is useful due to its strong fluorescent character.

BBa_K2657003: RCP Phytobrick

In order to improve a part already in the Biobrick Registry, we chose BBa_E1010, which expresses the red chromoprotein. We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor BBa_P10500 via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly reactions.

BBa_K2657004: RCP Phytobrick with a Strong Promoter

For this improvement, we took BBa_E1010 (RCP) and added the synthetic, strong constitutive promoter CP25 which functions in a broad range of organisms. This will increase the expression of the RCP. We also made the sequence golden gate compatible by inserting the sequence into the PhytoBrick Universal Acceptor BBa_P10500. By linking a Promoter/RBS with a coding sequence, the transformation efficiency of Golden Gate Assembly reactions will increase since the number of parts in the assembly will be reduced. This will also allow BBa_E1010 to be a more useful selective marker since, in its current form, it only expresses weakly and requires one to two days to express at all.