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Gluconobacter gene traceless knockout system and application

A gluconic acid bacillus, traceless knockout technology, applied in the field of genetic engineering, can solve the problems of immature genetic engineering operation technology, unsuitable for wide application, corrosiveness, etc., to reduce harmful risks, wide application range, and improve positive rate effect

Inactive Publication Date: 2015-11-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In 2005, the genome sequence of Gluconobacter oxydans 621H was published in Nat Biotechnol, 23(2), 195-200, which facilitated the research of genomics, proteomics and metabolomics. However, at present, the genetic engineering operation technology for Gluconobacter is not enough Mature
The limitation of this method is at first to obtain the upp gene-deficient bacterial strain, lacks universality; And the reverse screening object 5-fluorouracil itself belongs to poisonous substance, and danger level is Xn, median lethal dose (rat, oral) 230mg / kg , and is corrosive, not suitable for wide application

Method used

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  • Gluconobacter gene traceless knockout system and application
  • Gluconobacter gene traceless knockout system and application
  • Gluconobacter gene traceless knockout system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of the new integrated plasmid pJKM

[0043] Primers were designed according to the plasmid pSM20 (the base sequences are shown in SEQ ID NO.2 and SEQ ID NO.3), PCR amplification was used to obtain the SacB gene fragment, and the blunt-end cloning kit (pEASY-Blunt Simple Cloning Kit, full gold , Beijing) for blunt-end seamless cloning to obtain the recombinant plasmid pEASY-Blunt-SacB containing the SacB gene.

[0044] After the sequencing is correct, the SacB gene fragment is recovered by single-enzyme digestion with SspI, and connected with the pK18mobGII plasmid that has been digested with SspI, and transformed into E.coli DH5α. Colony PCR screening obtains positive clones with the correct connection direction of SacB, and the constructed integrated suicide Plasmid pJKM, see the plasmid map figure 1 shown.

Embodiment 2

[0045] Knockout of gluconate-2-dehydrogenase gene in Gluconobacter oxydans DSM2343 in embodiment 2

[0046] Gluconate-2-dehydrogenase (GOX1231) in Gluconobacter oxydans DSM2343 is a membrane-bound dehydrogenase with FAD as a coenzyme, which catalyzes the dehydrogenation of gluconate C2 to form 2 - Keto-D-gluconic acid.

[0047] According to the genome sequence (NC_006677) of Gluconobacter oxydans 621H in GenBank, the primer sequences were designed as follows:

[0048] 1231_HindIII_F: 5'-ataAAGCTTagccaaaggcggaaagacggc-3' (SEQ ID NO.4)

[0049] 1231_Fus_R: 5'-catttcaggggagaccgcttaaatgaagtggccgctggtcatc-3' (SEQ ID NO. 5);

[0050] 1231_Fus_F: 5'-gatgaccagcggccacttcatttaagcggtctcccctgaaatg-3' (SEQ ID NO. 6)

[0051] 1231_XbaI_R: 5'-ataTCTAGAcgccggcactttcttctacc-3' (SEQ ID NO. 7).

[0052] Using the above two pairs of primer sequences respectively, PCR was used to obtain 1000 bp of the upstream and downstream gene fragments of the GOX1231 gene, and by fusion PCR (with the upper...

Embodiment 3

[0055] Example 3 Knockout of pyruvate decarboxylase gene in G.oxydans ΔGOX1231

[0056] Pyruvate decarboxylase (GOX1081) is a key enzyme in the acetate pathway in Gluconobacter oxidans, which catalyzes the decarboxylation of pyruvate to form acetaldehyde and further to acetate. According to the genome sequence (NC_006677) of Gluconobacter oxydans 621H in GenBank, the primer sequences were designed as follows:

[0057] 1081_HindIII_F: 5'-cccAAGCTTctcgtctgggcgattcatg-3' (SEQ ID NO.8)

[0058] 1081_Fus_R: 5'-Cctgaggtactgaaatcatgacaaagcgtctgatccttcc-3' (SEQ ID NO.9)

[0059] 1081_Fus_F: 5'-Ggaaggatcagacgctttgtcatgatttcagtacctcagg-3' (SEQ ID NO.10)

[0060] 1081_SalI_R: 5'-acgcGTCGACAggcatgagacctacctga-3' (SEQ ID NO.11)

[0061] 800 bp of the upstream and downstream gene fragments of GOX1081 were obtained by PCR, and fused into the integrated plasmid pJKM to construct the suicide knockout plasmid pΔGOX1081. The suicide knockout plasmid pΔGOX1081 was electrotransformed into G.ox...

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Abstract

The invention discloses a gluconobacter gene traceless operation system and application. The gene traceless knockout system comprises a strain of wild gluconobacter and a recombined integrated suicide plasmid with SacB genes, antibiotic marks and multiple cloning sites. A method for knocking out genes in the gluconobacter through the gene traceless knockout system includes the steps that 1, the multiple cloning sites of the recombined integrated suicide plasmid are led into upstream and downstream segments of target genes to be knocked out, and a suicide knockout plasmid is obtained; 2, the obtained suicide knockout plasmid is transformed into the wild gluconobacter, antibiotic resistance screening and sucrose screening are sequentially performed, and gene defect strains with the target genes to be knocked out are obtained through colony PCR verification. The SacB genes are adopted as screening marks, gene traceless operation is performed on the gluconobacter, and the system is an improvement on a system with upp genes adopted as screening marks; in addition, no operation needs to be performed on host bacteria, the wild gluconobacter can be adopted, and the system is wide in application range and safe in operation.

Description

technical field [0001] The invention relates to a system for traceless operation of Gluconobacter gene, especially a system suitable for gene traceless knockout or traceless integration, and belongs to the technical field of genetic engineering. Background technique [0002] The genus Gluconobacter is a group of extremely flagellated, motile or non-motile bacterial populations, belonging to the Proteobacteria (Proteobacteria), α-proteobacteria (Alpha-proteobacteria), Rhodospirilla in taxonomic status Order (Rhodospirillales), Acetobacteraceae (Acetobacteraceae). It is an obligate aerobic Gram-negative bacterium, non-pathogenic to humans and animals, but it can cause bacterial rot and browning of fruits, so it is often found in sugar-rich environments, such as flowers, fruits, etc. . The strain contains a variety of membrane-bound dehydrogenases, such as D-glucose dehydrogenase (glucose dehydrogenase), glycerol dehydrogenase (glycerol dehydrogenase), D-sorbitol dehydrogenas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
Inventor 林建平袁建锋朱力吴绵斌杨立荣
Owner ZHEJIANG UNIV
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