Recombineering-mediated gene knockout method of corynebacterium glutamicum ATCC 13032

A technology of ATCC13032 and Corynebacterium glutamicum, which is applied in the field of genetic engineering, can solve the problems of time-consuming, laborious, tedious, and increasing difficulty, and achieve the effect of simple and fast method

Inactive Publication Date: 2015-04-29
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

It can be seen that this classic gene knockout method is time-consuming, laborious (requires multiple gene cloning and sequencing steps), and cumbersome (requires the participation of multiple strains and multiple experimental steps), because the final screening can simultaneously obtain the original strain For some genes, the gene knockout efficiency is often low, and a large number of clones need to be screened to obtain the target gene knockout mutant, which increases the difficulty of the experiment

Method used

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  • Recombineering-mediated gene knockout method of corynebacterium glutamicum ATCC 13032
  • Recombineering-mediated gene knockout method of corynebacterium glutamicum ATCC 13032
  • Recombineering-mediated gene knockout method of corynebacterium glutamicum ATCC 13032

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction of a plasmid that induces expression of the recombinase gene

[0044] A stuffer fragment containing the ribosome binding site is first cloned. Design primer R1407: 5'-GGG CTGCAG AAGGAGATATAGAT CCATGG CCTTCACCAGCACCTTGGTG-3', (SEQ ID NO.1), restriction sites PstI and NcoI are underlined, and the sequence between PstI and NcoI in italics is the ribosome binding site sequence, which is the gene cloned on the plasmid Required for expression in Corynebacterium glutamicum ATCC 13032; R1408: 5'-GGG GAATTC ACCCCCGGCCAGGCCAACTAC-3', (SEQ ID NO.2), the restriction site BamH is underlined. Using pSN101 as a template, R1428 and R1409 PCR to obtain a 1.5kb fragment, 1.5kb was digested with PstI and BamHI, and 3.0kb was ligated with pBluescript II KS(-) digested with the same enzyme, and transformed into Escherichia coli (Escherichia coli) DH10B. Transform competent cells in the presence of isopropyl-β-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-ind...

Embodiment 2

[0047] Example 2. Knockout of the crtI2 gene on the genome of Corynebacterium glutamicum ATCC 13032

[0048] 1. Preparation of Corynebacterium glutamicum ATCC 13032 electroporation competent cells and transformation of pLS2406

[0049] Corynebacterium glutamicum ATCC 13032 was streaked from a -80°C cryopreservation tube to BHIS solid medium, and cultured at 30°C for 24 hours. Transfer a single colony to 3ml BHIS liquid medium, culture at 30°C and 220rpm for about 24 hours, then transfer 2ml to 100ml BHIS, adjust the OD600 to about ~0.1, continue to cultivate until the OD600 is about 0.6, centrifuge, and discard the supernatant. The cell pellet was gently washed three times with ice-cold 10% glycerol, and finally suspended in 200 μl of ice-cold 10% glycerol, and each 50 μl was dispensed into an Eppendorf tube. Add 100ng of pLS2303 plasmid DNA to the competent cells, flick and mix well, transfer to a 2mm electric cuvette cooled on ice, and transform with 2.5kV electric shock. T...

Embodiment 3

[0059] Example 3. Knockout of the upp gene on the genome of Corynebacterium glutamicum ATCC 13032

[0060] Three pairs of primers R1431 were designed according to the sequence of the upp gene on the genome of Corynebacterium glutamicum ATCC 13032 and the sequence of the kanamycin resistance gene: 5'-ATAAAAGGGTGGTGGGGTTGGC-3', (SEQ ID NO.15), R1432: 5' - GCGCGGAACCCCTATTTGTTCATAGCTTCACATGTTAAATCATTGC-3', (SEQ ID NO. 16), R1433: 5'-GCAATGATTTAACATGTGAAGCTATGAACAAATAGGGGTTCCGCGC-3', (SEQ ID NO. 17), R1434: 5'-CCGTAATGCCCTTAGAAACTTTTATCCTTAGTTCCTATTCCGAAG, IDNO. R1435: 5'-CTTCGGAATAGGAACTAAGGATAAAAGTTTCTAAGGGCATTACGG-3', (SEQ ID NO. 19), R1436: 5'-GGAGGATGAAGTCCAAAGCTG-3', (SEQ ID NO. 20). Among them, R1432 and R1433, R1434 and R1435 are all reverse complementary to each other, and are used for amplification of overlap-extension PCR.

[0061] Using Corynebacterium glutamicum ATCC 13032 genomic DNA as a template, R1431 and R1432 PCR amplified to obtain a fragment of 500 bp upstrea...

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Abstract

The invention relates to a recombineering-mediated gene knockout method of corynebacterium glutamicum ATCC 13032. The gene knockout method comprises the following specific implementation steps: obtaining a DNA fragment which is provided with 500-bp homologous sequences on two sides aiming at genes to be knocked out and a kanamycin resistance gene in the middle through a polymerase chain reaction amplification; carrying out electrotransformation on the DNA fragment into a corynebacterium glutamicum ATCC 13032 cell in which recombinase is induced to express by isopropyl-Beta-D-thiogalactopyranoside, and enabling the kanamycin resistance gene to replace the target gene through the resistance selection of kanamycin to obtain gene knockout strains; finally, cultivating mutant strains in a solid medium containing cane sugar to eliminate plasmids containing recombinase genes. The gene knockout method adopts simple PCR (Polymerase Chain Reaction) and electrotransformation supplemented by the resistance selection of kanamycin, is free of operating steps, such as gene cloning in molecular biology and other certain operating steps, and simple and rapid, and has important application in the aspects of researching gene functions and producing amino acid.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a gene knockout method of Corynebacterium glutamicum ATCC 13032 mediated by recombinant engineering. Background technique [0002] Amino acids are essential substances for human growth and nutrition. Amino acids are widely used in industry, medicine and health and other fields. The annual output of amino acids is thousands of tons, and the amino acids produced on a large scale are all obtained through microbial fermentation. Corynebacterium glutamicum is the main amino acid producing bacteria. Most of the amino acid producing bacteria currently used in industrial production are derived from the original strain of Corynebacterium glutamicum ATCC 13032 through physical mutagenesis, chemical mutagenesis or genetic engineering. obtained by means. [0003] At present, the output of amino acids is on the rise, but it still cannot meet the requirements, and the amino acids that can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N15/66C12R1/15
Inventor 尚广东骆希凌文
Owner NANJING NORMAL UNIVERSITY
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