BBa_K2657005

This Phytobrick is an improved version of the Biobrick BBa_K864100, which is the coding sequence for super yellow fluorescent protein (sYFP2). We added overall usability and stability to this sequence by not only turning it into level 0 Phytobrick for use in Golden Gate Assembly, but we removed mutational hotspot from the sequence, making it more stable.

Originally, BBa_K864100 had the IS10 sequence: 5' - gctnagc - 3', which resulted in an insertion in the region of: 5’ -acaggcgtagtaccg- 3’. In BBa_xxxxxxx (the improved part), the IS10 sequence has been removed and the area of the insertion sequence is now 5’ -actggggttgttcca- 3’, which reduces the palindromic nature of the sequence. Palindromes in the DNA cause potential hairpins, which prevents RNA polymerase from translating the DNA correctly. We also turned this part into a level 0 Phytobrick by assembling it with the Universal Acceptor, BBa_P10500. This in turn will allow researchers to use this very fluorescent protein gene in Golden Gate Assembly reactions using the enzyme BsaI.

BBa_K2657003

In order to improve a part already in the Biobrick Regisry, we chose the Red Chromoprotein-expressing, BBa_E1010. 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

For this improvement, we took BBa_E1010 (RCP) and added the synthetic,strong constitutive promoter, CP25,. 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, for future use in Golden Gate Assemblies. By linking a Promoter/RBS with a coding sequence, this will increase the efficiency of the BsaI reaction in order to make transcriptional units in Golden Gate Assemblies.