Difference between revisions of "Part:BBa K4281003"

Latest revision as of 05:31, 26 September 2022

M271-14685-14690-up-M271-14685-14690-down

M271-14685-14690-up-M271-14685-14690-down

Contribution

Organ transplantation is the best choice for patients with organ failure. The number of kidney transplants in my country ranks second in the world. About 300,000 patients need organ transplantation each year. However, immune exclusion reactions will affect the long-term survival of transplant organs. The use of immunosuppressive agents can prevent immune rejection so that the long-term survival of transplant organs can reduce their adverse reactions to ensure the long-term high-quality life of transplant recipients. Rapamycin is a widely used clinical drug for the treatment of immune rejection, which can greatly improve the survival rate of transplanted organs after surgery. The traditional physical and chemical mutagenesis screening and fermentation process optimization enable the fermentation level of rapamycin to be obtained. Rapamycin is a new type of macrolide antibiotic, and it is a compound isolated from Streptomyces rapamycinicus through its antifungal activity that inhibits the growth of Candida albicans, Cryptococcus neoformans, Penicillium, and Mucosococcus. Because of its complex chemical structure, it is difficult to synthesize it by chemical methods, so this medicine is low productivity and it is also expensive. However, there are few studies on improving the fermentation yield of rapamycin through metabolic engineering. We tried to improve the fermentation yield of rapamycin by modifying the two-component signal transduction system in Streptomyces rapamycinicus. The two-component system is a basic control system for organisms to sense external stimuli and regulate various physiological metabolism and cell behaviors (Figure 1). It consists of histidine kinases and response regulatory proteins. The main type of signal transduction system is used. The two-component system is important for primary and secondary metabolism, morphological differentiation, osmotic pressure, and cell wall integrity of Streptomyces rapamycinicus.

Figure 1. Two-component system schematic diagram..

Engineering Success

To construct the engineered strain, we amplified the upstream and downstream homologous arm of gene M271_14685/M271_14690, cloned it into pKC1139 plasmid, and then transfer it into ET12567/pUZ8002 competent cell. Screen the correct strain and co-culture with Streptomyces rapamycinicus, choose the double cross-over strain and test the fermentation yield of rapamycin by HPLC.

I. Construction of double knockout plasmid experiment

1. PCR amplification of upstream and downstream homology arms gene of M271_14685/M271_14690.

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 2), double-enzyme digestion, and ligase to pKC1139 carrier.

Figure 2. Gel electrophoresis diagram.Line 1,3,5 are M271_14685/14690 upstream homology arm gene, 1208bp.Line 2,4,6 are M271_14685/14690 downstream homology arm gene, 1144bp...

In Figure 2, 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.

2. Gel electrophoresis of recombinant plasmid pKC-M271_14685/M271_14690.

Figure 3. Gel electrophoresis diagram.Line 1,3,5: recombinant plasmid pKC-M271_14685/ M271_14690, 10320bp.Line 2,4,6: recombinant plasmid pKC-M271_14685/ M271_14690 digested with EcoRI/HindIII, 7962 bp, 2358 bp...

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.

3. Recombinant plasmid pKC-M271_14685/ M271_14690 sequencing analysis.

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 (Figure 4-6.), and the plasmid was successfully constructed.

Figure 4. Global sequence alignment of sequencing..

Figure 5. The details of pKC1139-F sequence alignment..

Figure 6. 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.

4. Gel electrophoresis of single cross-over strains.

Figure 7. Gel electrophoresis diagram.Line 1: single cross-over strain-1, incorrect. Line 2: single cross-over strain-2, 1575bp, correct..

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 7).

5. Gel electrophoresis of double cross-over strains.

Figure 8. Gel electrophoresis diagram. Line 1: double cross-over strain,326bp, correct. Line 2: control strain: Streptomyces rapamycinicus NRRL 5491, 3475bp..

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 8), 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.

II. Functional test

 HPLC profiles of the wild-type strain Streptomyces rapamycinicus NRRL 5491 and the ΔM271_14685/M271_14690 mutant.

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Figure 9. 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 9 that the △M271_14685/m271_14690 detected more rapamycin at the same time as standard rapamycin.

6. Analysis of rapamycin production.

Figure 10. 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.

Sequence and Features