Engineering

His-ATUGT + EGFP

UGT is an enzyme catalyzing kaempferol into astragalin, and is the largest one among the three enzymes we used in our pathway (F3H, FLS, UGT), so we firstly constructed a His-ATUGT + EGFP vector to test the expression of UGT gene.

Protocol we used:

1. Verification of the sequence. The sequence we submitted here was come from (Arabidopsisthaliana). In order to get better field, we has done codon optimization for E.coli expression for this sequence. We contacted with a biology company to synthesize the sequence.
2. We constructed it into a EGFP tagged plasmid and transformed the plasmids into E. coli BL21(DE3).
3. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shook at 37℃ until OD = 600.
4. 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.
5. 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 a band at around 52.04kD, which is in line with the molecular weight of UGT. This band only occurred in IPTG group, which confirmed with our induction. Besides, UGT expression at 12 h was higher than that at 36 h. We proposed that this may be due to the large number of bacteria at 36 h and the high survival pressure, which is not conducive to protein expression. Taken together, we have successfully expressed UGT in E.coli.

AtF3H+AtFLS

F3H is an enzyme of 41.36 kD, which catalyzes naringenin into dihydrokaempferol. FLS is of 39.41 kD to catalyze dihydrokaempferol into kaempferol, we constructed an intact plasmid to express AtF3H and AtFLS respectively.

Protocol we used:

1. Verification of the sequence. The sequence we submitted here was come from (Citrus sinensis, Citrus unshiu). In order to get better field, we has done codon optimization for E.coli expression for this sequence. We contacted with a biology company to synthesize the sequence.
2. We constructed it into a pETDuet plasmid and transformed the plasmids into E. coli BL21(DE3).
3. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shook at 37℃ until OD = 600.
4. 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.
5. 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 41.36 kD and 39.41 kD, which are in line with the molecular weight of F3H and FLS. 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 were slightly higher than that with 36 h induction. Taken together, we have successfully expressed FSH and FLS in E.coli.

F3H-FLS+UGT

The final try is to construct a big plasmid consisting of three genes we have introduced to produce astragalin. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, F3H-FLS +UGT expression vector was constructed.

Protocol we used:

1. We constructed it into a pETDuet 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.