Colibacillus-corynebacterium inducible expression carrier pDXW-8 and building method thereof

A coryneform bacterium, induced expression technology, applied in the field of microbial genetic engineering, can solve the problems that the promoter cannot perform the transcription function very effectively, the tac promoter cannot be strictly controlled, etc.

Active Publication Date: 2010-04-14
JIANGNAN UNIV
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Problems solved by technology

All of these vectors have two disadvantages: one is that they contain fewer single restriction enzyme sites at the multiple cloning site, and the other is that the gene is expressed constitutively or in lacI q Inducible express

Method used

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  • Colibacillus-corynebacterium inducible expression carrier pDXW-8 and building method thereof
  • Colibacillus-corynebacterium inducible expression carrier pDXW-8 and building method thereof
  • Colibacillus-corynebacterium inducible expression carrier pDXW-8 and building method thereof

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Embodiment 1

[0029] The construction of embodiment 1 expression vector pDXW-8

[0030] The construction process of the expression vector pDXW-8 is as follows: figure 1 shown. We used the Escherichia coli expression vector pKK-223-3 as the mother plasmid for constructing the target vector. pKK-223-3 carries the tac promoter and the rrnBT1T2 terminator, both of which can effectively function in coryneform bacteria (Srivastava and Deb, 2005). In Escherichia coli JM109 and coryneform bacteria, in the presence of the inducer IPTG, the LacI repressor protein can be inactivated, and the target gene carried by pKK-223-3 can be induced to express.

[0031]Since coryneform bacteria are not sensitive to ampicillin, we introduced the kanamycin resistance gene kan into pKK223-3 as a selectable marker in the coryneform bacteria pair. Using plasmid pET-28a as a template and kan-F and kan-R as primers, a 1023bp kanamycin resistance gene was amplified by PCR. The restriction enzyme SgrAI digestion sit...

Embodiment 2

[0041] Example 2 Applicability Evaluation of Plasmid pDXW-8 in Escherichia coli and Brevibacterium flavum

[0042] To demonstrate the availability of pDXW-8, we expressed the vhb gene in E. coli and Brevibacterium flavum. The open reading frame of 441bp vhb gene was amplified by PCR using plasmid pMD-18-T-vhb as template and primers vhb-F and vhb-R with SD sequence as primers. Restriction enzymes EcoRI and HindIII digestion sites were introduced at the 5' and 3' ends of the amplified product. The PCR product was double cut with EcoRI and HindIII, and connected to pDXW-8 which was also double cut with EcoRI and HindIII to generate recombinant plasmid pDXW-8-vhb.

[0043] 1 mL of overnight cultured Escherichia coli JM109 was inoculated into 30 mL of LB medium, and JM109 (pDXW-8) and JM109 (pDXW-8-vhb) were respectively inoculated into 30 mL of LB medium containing kanamycin. Similarly, 1 mL of Brevibacterium flavum B.flavum cultured overnight was inoculated into 30 mL of LBG m...

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Abstract

A colibacillus-corynebacterium inducible expression carrier pDXW-8 and a building method thereof belong to the technical field of genetic engineering. The inducible expression carrier pDXW-8 can be copied and stably exists in colibacillus and corynebacterium and comprises a tac promoter, a terminator rrnBT1T2, a resistance marker card kanamycin mark resistance gene, a negative regulation gene laclPE104 and a multi-cloning site, wherein the tac promoter plays a transcript promoting function in the colibacillus and the corynebacterium, the terminator rrnBT1T2 has a transcript stopping function, the resistance marker card kanamycin mark resistance gene is used for selecting transformants of the corynebacterium, the negative regulation gene is used for severely controlling expression of the tac promoter, and the multi-cloning site includes 11 signal limited incision enzyme sites. In the corynebacterium, the carrier is moderate in foreign protein expression, and is particularly adaptable to study on corynebacterium metabolic engineering.

Description

technical field [0001] An Escherichia coli-coryneform bacterium shuttle induction expression vector pDXW-8 and its construction method belong to the field of microbial genetic engineering. Background technique [0002] Corynebacteria are a class of Gram-positive bacteria belonging to the actinomycetes with moderate to high GC content. Since Corynebacterium glutamicum was first isolated to produce L-glutamic acid (Kinoshita et al., 1957), three major representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum have been identified. Widely used in the production of amino acids. In particular, it is pointed out that there are only small differences among the three different species by DNA-DNA hybridization experiments (Liebl et al., 1991). [0003] Due to the accumulated knowledge about the physiology, biochemistry and genetics of C. glutamicum (Eggeling and Bott, 2005; Burkovski, 2008), metabolic engineering has rep...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12R1/15
Inventor 王小元徐大庆李烨谭延振
Owner JIANGNAN UNIV
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