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Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination

A technology of Pediococcus lactis and marker-free genes, applied in the field of marker-free gene knockout of Pediococcus lactis DQ2 based on homologous recombination, to achieve the effect of safe large-scale industrial production

Inactive Publication Date: 2016-08-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there is no report of gene knockout of Pediococcus lactis, so it is necessary to establish a knockout method of Pediococcus lactis

Method used

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  • Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination
  • Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination
  • Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Construction of pSET4E plasmid

[0047]The thermosensitive suicide plasmid pSET4S is an Escherichia coli-Gram-positive bacterial wide host range shuttle plasmid. The source of this plasmid is found in DaisuKeTaKamatsu, MaKotoOsaKi, and TsutomuSeKizaKi, ThermosensitiveSuicideVectorsforGeneReplacementinStreptococcussuis, Plasmid, 2001, 46(2): 140-148. The plasmid can replicate when the culture temperature is 37°C in Escherichia coli, so as to facilitate the cloning of homologous fragments, but cannot replicate when the culture temperature is 37°C in Streptococcus suis. This plasmid can be used not only for gene knockout of Streptococcus suis, but also for gene knockout of some other Gram-positive bacteria, so consider using this plasmid as an initial plasmid to construct a suitable knockout plasmid for gene knockout of P.acidilactici remove.

[0048] It was found that P.acidilacticiDQ2 was highly resistant to spectinomycin (>150μg / mL), but sensitive to eryt...

Embodiment 2

[0056] Example 2: Unmarked Knockout of P.acidilacticiDQ2ldh Gene

[0057] (1) Construction of ldh gene knockout plasmid pSET4E-Δldh

[0058] According to the upstream and downstream DNA sequences of the ldh gene (NCBI-GI=304328039) in the model strain DSM20284 of Pediococcus acidilactici, design corresponding primers up-F-ldh, up-R-ldh, down-F-ldh, down-R-ldh (SEQ ID NO : 7-10) PCR amplification ldh gene upstream and downstream sequences up-ldh (the sequence of about 1 kb size upstream of L-lactate dehydrogenase gene initiation codon) (SEQ ID NO: 11), down-ldh (L-lactate dehydrogenase A sequence of about 1 kb in size downstream of the stop codon of the hydrogenase gene) (SEQ ID NO: 12).

[0059] Using the genomic DNA of P.acidilacticiDQ2 as a template, the upstream and downstream homologous sequences of the ldh gene up-ldh, up-ldh, Down-ldh, the PCR amplification system is the same as before.

[0060] The PCR amplification conditions were as follows: pre-denaturation at 94°...

Embodiment 3

[0068] Example 3: Unmarked Knockout of the P.acidilacticiDQ2ldhD Gene

[0069] (1) Construction of ldhD gene knockout plasmid pSET4E-ΔldhD

[0070] According to the upstream and downstream DNA sequences of the ldhD gene (NCBI-GI=304329050) in Pediococcus acidilacticiDSM20284, design corresponding primers up-F-ldhD, up-R-ldhD, down-F-ldhD, down-R-ldhD (SEQ ID NO: 20-23) PCR amplification of its upstream and downstream sequences up-ldhD (the sequence of about 1 kb size upstream of the D-lactate dehydrogenase gene start codon) (SEQ ID NO: 24), down-ldhD (D-lactate dehydrogenase gene stop codon downstream approximately 1 kb sequence) (SEQ ID NO: 25).

[0071] Using the genomic DNA of P.acidilacticiDQ2 as a template, PCR cloned the upstream and downstream homologous sequences of the ldhD gene up-ldhD and For down-ldhD, the PCR amplification system remains the same as before.

[0072]The PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturatio...

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Abstract

The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.

Description

technical field [0001] The present invention relates to a gene knockout method based on the principle of homologous recombination, in particular to a marker-free gene knockout method for Pediococcus lactis DQ2 based on homologous recombination and a method prepared by knocking out ldh and ldhD genes of Pediococcus lactis DQ2 A strain producing optically pure D-lactic acid and L-lactic acid. Background technique [0002] Pediococcus acidilactici (P. acidilactici for short) is a kind of facultative anaerobic Gram-positive bacteria, which belongs to a kind of lactic acid bacteria. It performs homolactic acid fermentation and can tolerate very low pH value. Can also grow under osmotic pressure. The probiotic potential of this species of bacteria, the production of lactic acid and pediocin inhibit the growth of other microorganisms, and no toxic effects have been found in the current published articles. [0003] Pediococcus lactis P.acidilacticiDQ2 (preservation number: CGMCCNO...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/53C12N1/21C12R1/01
Inventor 鲍杰张鹏涂毅高秋强张建
Owner EAST CHINA UNIV OF SCI & TECH
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