4CL

Adding to part 4-Coumaryl ligase - 4CL (BBa_K1033001) [iGEM14_TU_Darmstadt]
In this year’s iGEM competition, SHSBNU_China team focused on anthocyanins production, especially delphinidin. 4CL gene is one of the key gene in the synthetic pathway of delphinidin, which catalyzed phenylalanine into 4-coumaryl coenzyme A.
We synthesized the sequence and constructed into plasmid pETDuet.
After transforming to E.coli BL21 and induced by IPTG overnight at 16 degree Celsius at various concentrations (0.25 to 2 uM), we lysed the bacteria and performed SDS-PAGE and we confirmed 4CL enzyme to be expressed successfully. Judged by the thickness of the SDS-PAGE band width and darkness, concentrations of iPTG doesn’t play a critical role in the expression of 4CL enzyme.

J23109

Adding to BBa_J23109
This year, we also studied the expression of J23109 series promoters. According to the sequences submitted by previous teams, we constructed three plasmids, PSB1C3-J23109-EV, PSB1C3-J23109-GFP and PSB1C3-J23109-RFP, respectively. Based on this, the expression effect of constitutive promoter J23109 was detected.
We transformed the constructed plasmid into E.coli DH5α and saw colonies with different colors on the plate. Furthermore, we also saw different colors in the liquid system after shaking, which was confirmed in the bacteria precipitation later. Therefore, these results indicate that the J23109 constitutive promoter can initiate the expression of downstream genes in E.coli DH5α.