Team:Austin UTexas/Improve

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Improve

BBa_K2657004: RCP Phytobrick with a Strong Promoter

For this improved part, we modified BBa_E1010 and added the synthetic broad host range promoter, CP25, and an RBS sequence to our improved part. The broad host range promoter will be compatible with a higher number of bacterial strains. We also made the sequence functional in MoClo Assembly with BsaI by inserting the sequence into the PhytoBrick universal acceptor, a modified pSB1C3 backbone.

Figure 1. The black circle encloses the successful transformation of the RCP phytobrick BBa_K2657004. The strong red color is clearly visible.

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 (GGA) reactions.

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 had an IS10 insertion sequence site, which resulted in an insertion in the region of: 5’ -acaggcgtagtaccg- 3’. In BBa_K2657005 (the improved part), the IS10 sequence has been removed and the area of the insertion sequence is now 5’ -actggggttgttcca- 3’. This sequence maintains the codon identity of the original sequence.

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.

Figure 2. Colonies are non- fluorescent under blue light. White-circled colonies highlight what expected colonies look like with the PhytoBrick.