TOXIN MANUFACTURE
1. TOXIN PROTEIN:
To ensure the successful establishment 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 were found which showed the successful expression of the mCherry after being cultured in solid LB with 80mM/L arabinose 24 hours after plate coating.
Fig 3. Engineered bacteria mCherry on plates
Fig 4. Engineered bacteria mCherry in pipets
For further verification of the protein expression, we coomassie stained our product and saw obvious expression of targeted site of the 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
We first successfully PCR our two target gene—the metK & orfX, which shows its function in improving the expression of avermectin, shown as Fig 6. PCR products are indicated. (See Protocol)
Fig 6. Successful PCR product of metK and orfX.
We then constructed our target gene 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 backbone and target fragments are indicated. (See Protocol)
Fig 7. metK and orfX in TA clone plasmid PMD-19T
Fig 8. metK and orfX in conjugation plasmid PL96 and PL97
We finally transformed our gene from a donor strain—E.coli ET12567 (a methylation defective strain) and the recipient strain—S. avermitilis with efficiency of approximately 1e-6. (See 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%.
