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Construction method and application of a kind of mycobacterium genetically engineered bacteria

A technology of genetically engineered bacteria and mycobacteria, applied in the field of genetic engineering, can solve problems such as low efficiency and complicated steps

Active Publication Date: 2021-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For mycobacteria, the traditional homologous recombination technology still has disadvantages such as low efficiency and complicated steps, so the present invention intends to construct CRISPR / Cas technology in wild strain mycobacteria

Method used

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  • Construction method and application of a kind of mycobacterium genetically engineered bacteria
  • Construction method and application of a kind of mycobacterium genetically engineered bacteria
  • Construction method and application of a kind of mycobacterium genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] A method for preparing a mycobacterium LY-1 genome editing vector based on the CRISPR-Cas9 system;

[0030] Synthesize Cas9 protein expression cassette (SEQ ID No.1), sgRNA expression cassette (SEQ ID No.2), kstd3-sgRNA fragment (SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7);

[0031] (1) The synthetic Cas protein expression cassette T-A was cloned from the pMD19-T-simple vector to obtain the plasmid T-Cas9. Then use primer P1 (5'-gaaacagaattaattaagcttTGGAAATCTAGAGGTGACCACAAC-3', SEQ ID NO.17) and primer P2 (5'-caaaacagccaagctgaattcTTAGTCGCCACCCAGCTGG-3', SEQ ID NO.18) to amplify the fragment, and pass through 5'-cggaggaatcacttcgca- 3' (SEQ ID NO.16) was seamlessly spliced ​​into pMV261 digested by BamH I and EcoR I to obtain plasmid pMV261-Cas9;

[0032] (2) Use primer P3 (5'-CCCAAGCTTTCGCGGTGAAAGACATGTTAGTTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC-3', SEQ ID NO.19) and primer P4 (5'-CCCAAGCTTAAAAAACAAGTTGATAACGGACTAGCCTTATTT-3', SEQ ID NO.20) to amp...

Embodiment 2

[0045] Using the constructed CRISPR / Cas9-based gene editing system, on the basis of knocking out the gene kstd3, knockout editing of 17β-hydroxysteroid dehydrogenase (17β-hsd) and 3-sterone-9α- The gene of hydroxylase (kshA) was inserted and edited;

[0046] The gene sequence of the 17β-hydroxysteroid dehydrogenase is shown in SEQ-ID-NO.9.

[0047] Synthesized Cas9 protein expression cassette (SEQ ID No.1), sgRNA expression cassette (SEQ ID No.2), 17β-hsd-sgRNA fragment (SEQ ID No.10, SEQ ID No. 11. SEQ ID No.12, SEQ ID No.13, SEQ ID No.14);

[0048] (1) The synthetic Cas protein expression cassette T-A was cloned from the pMD19-T-simple vector to obtain the plasmid T-Cas9. Then use primer P1 (5'-gaaacagaattaattaagcttTGGAAATCTAGAGGTGACCACAAC-3') and primer P2 (5'-caaaacagccaagctgaattcTTAGTCGCCACCCAGCTGG-3') to amplify the fragment, and after gel recovery, seamlessly splice into pMV261 digested with BamH I and EcoR I to obtain plasmid pMVC261- Cas9;

[0049] (2) Use primer ...

Embodiment 3

[0062] Utilizing the constructed gene editing system based on CRISPR / Cas9, 3-sterone-Δ 1 Knockout editing of -dehydrogenase (kstd3) and 17β-hydroxysteroid dehydrogenase (17β-hsd).

[0063] The 3-sterone-Δ 1 - The gene sequence of the dehydrogenase is shown in SEQ-ID-NO.8.

[0064] The gene sequence of the 17β-hydroxysteroid dehydrogenase is shown in SEQ-ID-NO.9.

[0065] The specific experimental method refers to Example 2, and the sterol conversion experiment of the successfully edited mycobacterium LY-1 is verified. The culture conditions are 30°C, 120rpm, 168h, after extraction with ethyl acetate, vacuum drying and reconstitution, and determine the sterol by HPLC. As a result of the transformation reaction, the molar yield of 9α-OH-AD reaches 47%, which is 12% higher than that of wild mycobacterium LY-1, as shown in the attached image 3 , shown in experimental group 2.

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Abstract

The invention discloses a construction method and application of a mycobacterium genetically engineered bacterium, belonging to the technical field of genetic engineering. In the present invention, after the recombinant plasmid is transformed into mycobacterium LY-1 cells, the sgRNA will recognize a specific region on the genome and guide the Cas9 protein to bind to the target site. Under the action of the protein, a double strand is formed at the target site Most of the cells die because of the broken gap: the homologous repair sequence introduced with the recombinant plasmid is integrated into the gap through double exchange under the action of recombinase, and the cells with successful gene homologous repair survive and form gene loss. Artificially designed genotypes such as living and exogenous gene insertions. The CRISPR-Cas9 system constructed by the present invention has a knockout efficiency of 60% for the same gene, which is more convenient and efficient than the existing mycobacterial gene editing methods.

Description

technical field [0001] The invention relates to a construction method and application of a mycobacterium genetically engineered bacterium, belonging to the technical field of genetic engineering. Background technique [0002] Steroidal drugs have important physiological activities and are widely used clinically in the treatment of rheumatism, cardiovascular, collagenous diseases, lymphoid leukemia, human organ transplantation, anti-tumor, bacterial encephalitis, skin diseases, endocrine disorders, elderly Diseases, etc., are the second largest class of drugs next to antibiotics. Currently, the annual sales of steroid drugs worldwide exceed 100 billion US dollars. 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) is an important precursor of steroid hormones. Important precursors of hormones, such as dexamethasone, betamethasone, and mometasone furoate, have important commercial value and extensive market demand. [0003] Microbial fermentation produces sterols, which can simpl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P33/06C12R1/32
CPCC12N9/0004C12N9/0006C12P33/06C12Y101/01051
Inventor 许正宏李会史劲松许桠楠张晓梅龚劲松
Owner JIANGNAN UNIV
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