Continuous traceless gene knockout method for corynebacterium glutamicum

A Corynebacterium glutamicum, traceless knockout technology, applied in the field of genetic engineering, can solve the problems of cumbersome and lengthy knockout process and low homologous recombination, and achieve the effect of simple and effective screening process and time saving.

Active Publication Date: 2017-09-01
SOUTH CHINA UNIV OF TECH
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AI Technical Summary

Problems solved by technology

However, due to the limitation of the self-recombination system of Corynebacterium glutamicum, the probability of homologous recombination is very low without the assistance of an integrated plasmid
This makes the homologous recombination in Corynebacterium glutamicum need to be mediated by the integration type plasmid, and makes the whole knockout process cumbersome and lengthy

Method used

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  • Continuous traceless gene knockout method for corynebacterium glutamicum
  • Continuous traceless gene knockout method for corynebacterium glutamicum
  • Continuous traceless gene knockout method for corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Exonuclease-recombinase to the recombination of linear double-stranded DNA in Corynebacterium glutamicum

[0048] 1. Preparation of Corynebacterium glutamicum Competent Cells Expressed by Exonuclease-Recombinase

[0049] Design primers recT / recE-S: 5′-CAAAAAGGAGGCCCTTCAGATGAGCACAAAACCACTCTTC-3′ and recT / recE-A: 5′-GGCACCAATAACTGCCTTAATTATTCCTCTGAATTATCGA-3′ to amplify the gene recT+recE;

[0050] Design primers orfC / orfB-S: 5′-CAAAAAGGAGGCCCTTCAGATGACAAAATTATGTTTTAGTG-3′, orfC / orfB-A: 5′-GGCACCAATAACTGCCTTAATTAAGCCTTTATCCTGATTAGT-3′, amplify gene orfC+orfB;

[0051] Design primers orf48 / orf47-S: 5′-CAAAAAGGAGGCCCTTCAGATGGCTATTGCAAAAGAAAAGAC-3′, orf48 / orf47-A: 5′-GGCACCAATAACTGCCTTAATTAGATCATTGACCCTTGAACC-3′, amplify orf48+orf47 gene;

[0052] Design primers gp60 / 61-S: 5′-ACAAAAAGGAGGCCCTTCAGATGAGTGTGCCCACACAGGACGGA-3′, gp60 / 61-A: 5′-GGCACCAATAACTGCCTTAATCATGCGTTGGGCCCGTCGAACAT-3′, amplify gp60+gp61 gene;

[0053] Design primers PEC-A: 5'-CTGAAGGGCCTCCTTTTTGT...

Embodiment 2

[0066] Example 2 Obtaining of single gene traceless knockout strain ATCC14067-RecET-MargR

[0067] 1. Preparation of self-cut linear double-stranded DNA knockout expression cassette ΔArgR

[0068] Design primers PBS-Kan-S: 5'-TCGATCCTTTTTAACCCATTGCAGGAATTCGATATCAAG-3', PBS-Cre-A: 5'-GCGACACGAATTATGCAGTTTGTTATCCGCTCACAATTC-3', amplify the 2875bp plasmid pBluescript II SK (+) backbone fragment;

[0069] Primers theoE-Cre-S: 5′-ACTGCATAATTCGTGTCGCTCAAG-3′, theoE-Cre-A: 5′-CTTTGCGCTTGCGCGGAATTAATTCATGAGCG-3′ were designed to amplify the 1594bp inducible Cre enzyme expression cassette;

[0070] Design primer Kan-S: 5′-AATTAATTCCGCGCAAGCGCAAAGAGAAAGCA-3′,

[0071] Kan-A: 5′-ATGGGTTAAAAAGGATCGATCCTC-3′, amplified 1074bp Kan resistance screening marker expression cassette;

[0072] After recovering the above three fragment gels, they were assembled with the NEBuilder HiFi DNA Assembly Master Mix (NewEngland BioLabs) kit to obtain the universal plasmid PBS-Cre-Kan.

[0073] Universa...

Embodiment 3

[0080] Example 3 Superimposed traceless knockout of the crtB gene in the traceless knockout strain ATCC14067-RecET-MargR

[0081] Design primers crtB-L-S (1) according to the method in Example 2: 5'-GAAGTGTTGATAGAAATGACCTCA-3', crtB-L-loxp71: 5'-TGCAGTATAACTTCGTATAATGTATGCTATACGAACGGTAATGAAGACATCAACTACAACTCC-3', amplify 840 bp containing crtB using the genome of ATCC14067 as a template The DNA sequence of the left homology arm of the gene and the loxp71 sequence (C-1);

[0082] Design primers crtB-R-loxp66: 5′-ACCCATATAACTTCGTATAGCATACATTATACGAACGGTATCATAGCTGAGCCTGCTTCTGG-3′, crtB-R-A(1): 5′-GTCACTAGTGCTGACTCCCCCTCT-3′, amplify 850bp right homology arm containing loxp66 and crtB gene using ATCC14067 genome as template DNA sequence (C-3).

[0083] Using crtB-L-S(1), crtB-R-A(1) as primers, fragment C-1, 2712bp universal Cre-Kan expression cassette (fragment 2) and fragment C-3 as template, fusion 4322bp ΔCrtB self-cleavage A linear knockout expression cassette for .

[0084] A...

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Abstract

The invention discloses a continuous traceless gene knockout method for corynebacterium glutamicum and belongs to the technical field of gene engineering. The continuous traceless gene knockout method is characterized in that an exonuclease-recombinase-Cre/loxP continuous traceless knockout system, preferably an RecET-Cre/loxP continuous traceless knockout system, is built in corynebacterium glutamicum. According to the method, mediation of plasmids is not required by homologous recombination in corynebacterium glutamicum, and under assistance of RecET, transformation into linear double-stranded DNA is only required; only one round of transformation is required to finish traceless knockout of target genes, the whole traceless knockout process only needs 4-6 days, and meanwhile, continuous traceless gene knockout can be finished; and as a resistance gene is utilized as a selection marker, the selection process is simple and effective, and compared with other traceless knockout methods for corynebacterium glutamicum, the continuous traceless gene knockout method provided by the invention is simpler, more effective and more time-saving.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for continuously and seamlessly knocking out genes in Corynebacterium glutamicum. Background technique [0002] Corynebacterium glutamicum is widely used in the production of L-amino acids, vitamins, fuel ethanol and other organic acids, and is currently mainly completed in Corynebacterium glutamicum through metabolic engineering and synthetic biology methods The accumulation of corresponding metabolites, so the method of continuous traceless knockout is particularly important. In the model strain ATCC13032 of Corynebacterium glutamicum, methods such as reverse selection marker system, Cre-loxP recombination system, RecT-mediated single-stranded DNA recombination system and CRISPRi interference system have been successfully applied to the target gene without trace knockout In addition, point mutation and inhibition of target gene expression have laid a solid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12R1/15
Inventor 郑穗平黄园园林影韩双艳
Owner SOUTH CHINA UNIV OF TECH
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