Gluconobacter oxydans nonresistant marker gene knockout system

A Gluconobacter, no resistance marker technology, applied in the field of genetic engineering

Active Publication Date: 2014-05-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using the upp gene as a screening gene to knock out the gene in G.oxydans

Method used

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  • Gluconobacter oxydans nonresistant marker gene knockout system
  • Gluconobacter oxydans nonresistant marker gene knockout system
  • Gluconobacter oxydans nonresistant marker gene knockout system

Examples

Experimental program
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Effect test

Embodiment 1

[0018] The construction of embodiment 1 recombinant integration plasmid

[0019] The upp gene (SEQ ID NO.1, including promoter) annotated in the whole genome sequencing results of G.oxydansWSH-003 (preserved in the Industrial Microbiology Resource and Information Center of Chinese Universities, Jiangnan University, No. CICIM-CUB7004) in our laboratory was designed The primers were amplified, respectively connected to the cloning vector pMD19-T for sequencing, and after obtaining the correct upp transformants, they were double digested and connected to pK19mobsacB to construct pK19mobupp ( figure 1 ).

Embodiment 2

[0020] The construction of embodiment 2upp gene-deficient G.oxydans engineering bacteria

[0021] The primers were designed to amplify the 500bp upstream and downstream genes annotated in the whole genome sequencing results of G.oxydansWSH-003, and the 500bp genes upstream and downstream of the upp gene were spliced ​​by fusion PCR to obtain the upp gene knockout framework, which was connected to Sequencing of the cloning vector pMD19-T, after obtaining the correct transformants, double enzyme digestion was performed to obtain the upp gene knockout frame, electrotransformed into G.oxydansWSH-003, coated with sorbitol plates containing 5-fluorouracil, and positive transformants were verified by colony PCR (Only the upstream and downstream fragments of the upp gene, no upp gene), and the upp gene-deficient G.oxydansWSH-003 was obtained.

Embodiment 3

[0022] Knockout of sorbitol reductase gene in embodiment 3G.oxydansWSH-003

[0023] Connect the knockout framework of the Sorbose Reductase gene (upstream and downstream gene fusion fragment) to the MCS multiple cloning site of the constructed recombinant integration plasmid pK19mobupp, electrotransform the upp gene-deficient G.oxydansWSH-003, and coat On the sorbitol plate containing kanamycin, the grown transformant was transferred to the sorbitol plate containing 5-fluorouracil and thymine, and the positive transformant knocked out of the sorbitol reductase gene was obtained through PCR verification, only sr Upstream and downstream gene fragments, sr gene does not exist.

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Abstract

The invention discloses a gluconobacter oxydans nonresistant marker gene knockout system, and belongs to the technical field of genetic engineering. According to the invention, through a homologous recombination technique, upp genes in G.o xydans are knocked out, so that a upp genetic deficient strain is obtained; a recombinant integrating plasmid containing a upp gene which can be subjected to good transcribed expression in G.o xydans and has a conditional lethal function, a resistance maker kanamycin resistant marker gene for the transformant selection of G.o xydans and an MCS polycloning site is built. The plasmid is applicable to the knockout and replacement of G.o xydans chromosome genes; compared with gene knockout implemented by taking a resistant marker gene as a selection marker, the system disclosed by the invention has the following two advantages: no resistant gene is introduced to a G.o xydans genome, so that a polar effect of the resistant gene on upstream and downstream genes is avoided; by using the knockout system, the multiple knockout and replacement of chromosome genes can be performed in a same strain.

Description

technical field [0001] The invention relates to a non-resistance marker gene knockout system of Gluconobacter oxydans, in particular to a system for gene knockout in G.oxydans using upp gene as a screening gene and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Gluconobacter oxydans (Gluconobacteroxydans) is an obligate aerobic Gram-negative bacteria belonging to the family Acetobacteriaceae. It is non-pathogenic to humans and other animals, but it can cause bacterial rot and browning of fruits. The strain contains a variety of enzymes that can incompletely oxidize organic matter and produce a variety of important compounds, which makes it a place in industrial applications. There are two main types of enzymes: the first is granzyme, which is combined on the cell membrane, such as D-glucose dehydrogenase, glycerol dehydrogenase, D-sorbitol dehydrogenase, etc. These enzymes are flavoproteins or cytochrome...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/74
Inventor 陈坚周景文夏雨堵国成
Owner JIANGNAN UNIV
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