Part:BBa_K4236007:Experience

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Applications of BBa_K4236007

Protocol we used: 1. We constructed it into a (请老师确认载体骨架) plasmid and transformed the plasmids into E. coli BL21(DE3). 2. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shaken at 37℃ until OD = 600. 3. Dividing the liquid medium into 23 parts, adding 1 mM IPTG to two of them for inducing the expression 16℃ at 12 h or 36 h . The other one added nothing to serve as a control. 4. Centrifuged the bacterial solution at 12000 g, the precipitation was mixed with RIPA as a lysis buffer. Added loading buffer to heat at 96℃ for 10 min, underwent SDS-PAGE and Coomassie brilliant blue staining for expression test. According to our SDS-PAGE result, we could see two band at around 81 kD and 51kD, which are in line with the molecular weight of F3H-FLS fusion protein and UGT. The two band only occurred in IPTG group, which confirmed with our induction. in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction weas slightly higher than that with 36 h induction. Taken together, we have successfully expressed the whole pathway to produce astragalin in E.coli.

Fig. 1: The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT.)

Fig. 2: The exprssion test for CsF3H-CuFLS+AtUGT)

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