We designed the program by inserting the M271_14685/14690 upstream homology arm gene into HindIII and EcoRI sites of the pKC1139 vector. In order to build our plasmids, we amplified the gene fragments from the genome of Streptomyces rapamycinicus NRRL 5491 by PCR (Figure 1), double-enzyme digestion, and ligase to pKC1139 carrier.
Figure 1: Gel electrophoresis diagram. In Figure 1, a clear and single DNA band at 1kp can be seen, indicating that the upstream and downstream homology arms of M271_14685/M271_14690 were successfully amplified by PCR.
Figure 2:Gel electrophoresis diagram. After selecting the correct colony by colony PCR, we inoculated it in LB with the antibody and extract the plasmid. To verify if the plasmid is correct, we did double-enzyme-digestion. It can be seen from the figure that the size of the plasmid we constructed is correct, and the identification results of double enzyme digestion are also correct.
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure3-5.), and the plasmid was successfully constructed.
Figure 3: Global sequence alignment of sequencing.
Figure 4: The details of pKC1139-F sequence alignment.
Figure 5: The details of pKC1139-R sequence alignment.
As shown in the three figures, the sequencing results further demonstrated that the pKC-M271_14685/ M271_14690 construction was correct, and consistent with PCR identification results.
Figure 6: Gel electrophoresis diagram. To construct the engineering strain, we firstly transferred the recombinant plasmid into ET12567/pUZ8002 competent cells and screened the correct strain through 3 antibodies, and cultured it in the liquid medium. Co-cultured the E.coli with Streptomyces rapamycinicus and screened for the single cross-over strain.
We selected two strains to identify the single exchange of gene fragments, and the results showed that the single cross-over strain 2 was successful (Figure 6).
Figure 7: Gel electrophoresis diagram.Based on the screened single cross-over strain, we further subcultured and screened for the double cross-over strain, and that’s the engineering strain we needed. We also used colony PCR to verify (Figure 7), and as the figure shows, we construct the strain successfully. Compared with the negative control, the gene fragments of the engineered bacteria we constructed were successfully exchanged. That is, the engineered bacteria that knocked out the M271_14685/M271_14690 gene were successfully obtained, and we named the strain △M271_14685/m271_14690.
Figure 8: A is rapamycin, B is wild-type NRRL 5491, and C is
△M271_14685/m271_14690.
Co-cultured Streptomyces rapamycinicus NRRL 5491 with ΔM271_14685/M271_14690 in the fermentation medium, we collected the sample respectively at 5d,7d,9d, and 11d. We mixed 0.5 mL samples with the same volume of methanol and tested the yield of rapamycin after centrifuge. To identify the correct peek of rapamycin, we use the standard rapamycin as positive control. It can be seen from Figure 8 that the △M271_14685/m271_14690 detected more rapamycin at the same time as standard rapamycin.
Figure 9: zz △M271_14685/m271_14690 and NRRL 5491 have produced rapamycin during 11
days.
It can be seen from the above figure that the rapamycin produced by our knockout △M271_14685/m271_14690 is much higher than that produced by NRRL 5491. And the amount of rapamycin produced on the ninth day reached 120mg/L. This suggests that the subject is feasible to improve metabolic pathways by knocking out the two-component system, which can be applied for clinical application in the near future.
