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A Gluconobacter oxydans resistance-free marker gene knockout system

A technology of no resistance marker and gene knockout, applied in the field of genetic engineering

Active Publication Date: 2016-11-02
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 and the method of constructing the knockout system have not been reported in China

Method used

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  • A Gluconobacter oxydans resistance-free marker gene knockout system
  • A Gluconobacter oxydans resistance-free marker gene knockout system
  • A Gluconobacter oxydans resistance-free marker gene knockout system

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Experimental program
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Embodiment 1

[0018] The construction of embodiment 1 recombinant integration plasmid

[0019] The upp gene (SEQ ID NO.1, containing promoter) to design primers for amplification, respectively connected to the cloning vector pMD19-T for sequencing, after obtaining the correct upp transformant, perform double enzyme digestion and connect to pK19mobsacB, and 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.oxydans WSH-003, and the 500bp genes upstream and downstream of the upp gene were spliced ​​by fusion PCR to obtain the upp gene knockout framework, ligated Sequenced to 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.oxydans WSH-003, coated with sorbitol plate containing 5-fluorouracil, and the colony PCR was positive Transformants (only upp gene upstream and downstream fragments, no upp gene) were obtained upp gene-deficient G.oxydans WSH-003.

Embodiment 3

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

[0023] Connect the knockout framework of the Sorbose Reductase gene (upstream and downstream gene fusion fragments) to the MCS multiple cloning site of the constructed recombinant integration plasmid pK19mobupp, electrotransform upp gene-deficient G.oxydansWSH-003, and coat Put the sorbitol plate containing kanamycin, and 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. 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 (Gluconobacter oxydans) 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 cytochrom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/74
Inventor 周景文陈坚夏雨堵国成
Owner JIANGNAN UNIV
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