We have improved the function of three existing parts this year.
We added two orthogonal spacer sequences to part ‘J23101-GFP’ (BBa_J364000) and ‘J23101-RFP’ (BBa_K516132) respectively so that these parts could be regulated by corresponding sgRNAs (BBa_K2611000 and BBa_K2611002). We have confirmed that the modified promoters do not influence the original function of these parts. What’s more, co-transformed with dCas9 plasmid, the corresponding sgRNA could repress the fluorescent intensity in about 40% degree. In summary, we improved the regulatory property of these parts, which promise that the new parts (BBa_K2611001 and BBa_K2611003) could respond to our CRISProgrammer.
The other one is T7-RFP-J23100-GFP (BBa_K2611007) which containing two original reporter parts together in one vector. The two original parts are J23100-GFP (BBa_J364007) and T7-RFP (BBa_K199118). In this way, we improved the fluorescent properties of the original parts as the new part could express green and red fluorescence at the same time. This new part represents the final indigo synthesis pathway and is important in verifying the function of our CRISProgrammer.
Team Berkeley 2013 also focused on indigo synthesis. Instead of glucosyltransferase (GT), we chose UDP-glucuronyl transferase (UGT) which provides a higher catalytic efficiency. Also, we processed codon optimization of B-glucosidase enzyme (BGL) for its usage in E. coli. As for regulatory system, instead of chemical induction, we constructed CRISProgrammer system to modulate expression of UGT and BGL, which simplifies the regulatory process especially in industrial manufacture.