TOXIN MANUFACTURE
1. Toxin protein:
To confirm the successful construction of the toxin plasmids, we electrophoresed both the single and double digestion product of cloned mCherry (CDS), plu1537 (Device) and plu0840 (Device) shown as below.
Fig 1. Engineered toxin protein circuit-1 (single and double digestion)
Fig 2. Engineered toxin protein circuit-2 (double digestion)
Red colonies in plates indicated the successful expression of the mCherry after being cultured in LB plates with 80mM/L arabinose 24 hours.
Fig 3. Engineered bacteria mCherry on plates
Fig 4. Engineered bacteria mCherry in pipets
For further verification of the protein expression, we did SDS-PAGE and observed obvious expression of mCherry, plu1537 and plu0840 (see lane1, lane6 and lane7), but no obvious expression band was found for tcdA1 (see lane 8). From lane2 to lane5, we could only see significant result in lane4 where high expression of mCherry might occur.
Fig 5. Expression of our toxin protein
2. Avermectin overexpression
To overexpress the avermectin in Streptomyces avermitilis ATCC13267
We first successfully PCR our two target gene: the metK & orfX, both of which show their function in improving the productivity of avermectin, shown in Fig 6. More PCR parameters please go to Protocol.
Fig 6. Successful PCR product of metK and orfX.
Secondly, we constructed metK and orfX into TA clone plasmid PMD-19T (See fig 6) and then transformed them into our conjugation transferring plasmid PL96 and PL97 (see fig 7). Digested plasmid backbones and target fragments are indicated. More experimental parameters please go to Protocol.
Fig 7. metK and orfX in TA clone plasmid PMD-19T
Fig 8. metK and orfX in conjugation plasmid PL96 and PL97
Thirdly, we transformed our gene from a donor strain: E.coli ET12567 (a methylation defective strain) to the recipient strain: S. avermitilis with efficiency of 1e-6, approximately. More experimental parameters please go to Protocol.
Fig 9. Engineered S. avermitilis on plates
Finally, we succeed in getting the positive result of the Avermectin-enhanced S.A strain by plasmid PL97+orfX. The result shows that we change the average production of Avermectin (liquid bacteria OD600 0.5) of wildtype (2.361ug/ul) to 3.703(ug/ul). The ratio of enhancement is up to 56.81%, which indicates the comparatively high efficiency of improving avermectin productivity.
