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A kind of gene knockout method of Actinobacillus succinates

A technology of actinomycetes and succinic acid, applied in the field of genetic engineering and metabolic engineering transformation, can solve the problems of high cost, limit the wide application of basic chemical raw materials, environmental pollution, etc., achieve excellent production performance, high conversion efficiency, and promote research progress Effect

Active Publication Date: 2020-12-08
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The traditional chemical synthesis of succinic acid uses butane to be produced by electrolysis through maleic anhydride, which has problems such as high cost and environmental pollution, which limits its wide application as a basic chemical raw material

Method used

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  • A kind of gene knockout method of Actinobacillus succinates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This example specifically illustrates the construction method of the left and right homology arms of the pyruvate formate lyase gene pflB in Actinobacillus succinogenes NJ113, including the following steps:

[0027] (1) Design PCR amplification primers to construct left and right homology arms:

[0028] Left arm upstream primer: GACTAGTCTAGCGCAAGGCTAACGGTGCTTC.

[0029] Left arm downstream primer: ATAACGTCTTGTTGCTGTTCGCGTTCCTGCCATTCACCGGGAACGA.

[0030] Right arm upstream primer: TCGTTCCCGGTGAATGGCAGGAACGCGAACAGCAACAAGACGTTAT.

[0031] Right arm downstream primer: GACTAGTCGATACCGCGGCTTACATTACGGC.

[0032] (2) Use the upstream primer of the left arm and the downstream primer of the left arm to amplify the homologous left arm by PCR using the pyruvate formate lyase gene pflB in Actinobacillus succinogenes NJ113 as a template, and use the upstream primer of the right arm and the downstream primer of the right arm The homologous right arm was amplified by PCR using the p...

Embodiment 2

[0036] This example specifically illustrates the method for screening the correct positive monoclonal clones of the pflB left and right homologous arm-linked suicide plasmid pJC4:

[0037] Select SacⅠ as the enzyme cutting site to single-digest the suicide plasmid pJC4, use one-step cloning to link the left and right homologous arms of pflB to the suicide plasmid pJC4, take 20uL of the enzyme-linked reaction product, and add it to 100uL of the suicide plasmid donor bacteria Donor competent cells , mix well by flicking the tube wall a few times, place on ice for 30 minutes, heat shock at 42°C for 45-90 seconds, incubate in an ice-water bath for 2 minutes, add 900uL SOC or LB medium, shake the bacteria at 37°C for 45 minutes, centrifuge at 4500r / min for 3 minutes, discard The supernatant was 900uL, and 100uL of the resuspended bacteria was left and evenly spread on the LA plate with a screening concentration of chloramphenicol of 25ug / mL. Invert the plate and culture overnight a...

Embodiment 3

[0041] This example specifically illustrates the screening method for constructing the recombinant strain of Actinobacillus succinate NJ113△pflB:

[0042] (1) Take 100uL each of the overnight bacteria of Donor and Actinobacillus succinogenes NJ113, add to the same EP tube, mix well, and centrifuge at 4500r / min for 3min;

[0043] (2) Resuspend the mixed bacteria in (1) with 10uL LB, place the 10uL bacteria on the chloramphenicol-free LA plate coated with 2,6-diaminopimelic acid for 2-3 spots , after drying the bacterial solution, place it in an incubator at 37°C for more than 15 hours;

[0044] (3) Pick the plaque on the plate in (2) and wash it once with LB in the EP tube, resuspend it, apply it on an LA plate with a chloramphenicol screening concentration of 5ug / mL, and culture it in an incubator at 37°C until single cells grow on the plate. clone colonies;

[0045] (4) Pick the monoclonal colonies in (3) to LB for overnight culture, use 20% glycerol to preserve the seed, a...

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Abstract

The invention discloses an actinobacillus succinogenes gene knock-out method. The method comprises the following steps: constructing left and right homologous arms of target genes; constructing suicide plasmids with antibiotic screening markers and negative screening markers; connecting the left and right homologous arms of target genes with the suicide plasmids, transferring into donor strain competent cells, screening accurate positive mono-clones by virtue of resistant spread-plate, and performing sequencing validation; and transferring the accurate positive mono-clones into objective strains actinobacillus succinogenes, screening single-crossing-over positive mono-clones in the resistant spread-plate, and performing negative screening on the positive mono-clones subjected to accurate single-crossing-over, thereby obtaining the recombinant strain with target gene deletion. The method disclosed by the invention is specified at actinobacillus succinogenes gene knock-out genes, and issimple in operation, high in conversion rate, applicable to multi-gene knock-out and capable of realizing non-trace knock-out of the target genes.

Description

technical field [0001] The invention relates to the field of genetic engineering and metabolic engineering transformation, in particular to a high-efficiency gene knockout method for Actinobacillus succinate. Background technique [0002] Succinic acid (also known as succinic acid) is a common natural organic acid widely found in human body, animals, plants and microorganisms. As an important chemical raw material, it is also widely used in pharmaceutical, food and surfactant industries. Succinic acid can be used as an intermediate in the synthesis of complex organic compounds, and also as a C 4 Platform compounds are used in the synthesis of bulk chemicals and biodegradable materials, so succinic acid is considered to be the most marketable fermented organic acid product after citric acid. [0003] The traditional chemical synthesis of succinic acid is produced by electrolysis of butane through maleic anhydride, which has problems such as high cost and environmental pollu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/65C12R1/01
CPCC12N15/65C12N15/74
Inventor 姜岷朱君如信丰学章文明董维亮马江锋
Owner NANJING TECH UNIV
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