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Construction method and application of mycobacteria genetic engineering 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: 2020-02-14
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 mycobacteria genetic engineering bacteria
  • Construction method and application of mycobacteria genetic engineering bacteria
  • Construction method and application of mycobacteria genetic engineering 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 mycobacteria genetic engineering bacteria, and belongs to the technical field of genetic engineering. A recombinant plasmid is transformed into a mycobacteria LY-1 cell, sgRNA can distinguish a specific region on a genome and lead Cas9 protein to be combined to a target site; and under the action of the protein, a double-strand breaknotch is formed in the target site, and most of cells are dead. Under the action of recombinase, a homologous repairing sequence led along with the recombinant plasmid is integrated to the notch through double exchange, and the cells successfully repaired homologously by genes can survive, and form genotypes which are artificially designed for gene inactivation, exogenous gene insertion and the like. The knockout efficiency for the same gene by the constructed CRISPR-Cas9 system is 60%, and compared with a conventional mycobacteria gene editing manner, the constructed CRISPR-Cas9 system is more convenient and higher in efficacy.

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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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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