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Corynebacterium promoter detection vector and construction method and application thereof

A coryneform bacterium and promoter technology, applied in the field of microbial genetic engineering, can solve the problems of limited amino acid production, weak promoter activity, and low expression level of foreign genes

Active Publication Date: 2011-03-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, almost all the existing coryneform bacteria expression vectors use E. coli promoters such as lacUV5, trp, tac, λP R P L and araBAD promoters to express foreign genes (Srivastava and Deb, 2005); however, in coryneform bacteria, the activity of these promoters is weak, and the expression level of foreign genes under their control is usually not high, which limits amino acid production

Method used

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  • Corynebacterium promoter detection vector and construction method and application thereof
  • Corynebacterium promoter detection vector and construction method and application thereof
  • Corynebacterium promoter detection vector and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Construction of expression vector pDXW-11

[0029] The construction process of the expression vector pDXW-10 is as follows: figure 1 , as shown in 2.

[0030] In the first step, based on the expression vector pET28a (Novagen, USA), the lacI gene and the T7 promoter were removed to construct the plasmid pDXW-1 ( figure 1 ). The carrier pET28a was double-digested with restriction enzymes XbaI and BglI, the obtained large fragment was purified, and the large fragment was self-circularized with T4 ligase. The size of the constructed recombinant plasmid was 3524bp, named pDXW-1( figure 1 );

[0031] In the second step, construct the shuttle vector pDXW-2 ( figure 1 ). Taking the plasmid Escherichia coli-coryneform bacteria shuttle plasmid pC2 (Goyal et al., 1996Plasmid.36:62-66) as template, rep-F and rep-R as primers, the coryneform bacterium replicon fragment rep of 2686bp was amplified by PCR , A restriction enzyme SmaI restriction site was introduced at th...

Embodiment 2

[0040] Example 2 Application of the detection vector pDXW-11 to detect the promoter

[0041] In order to demonstrate the availability of pDXW-11 and search for promoters that can be highly expressed in coryneform bacteria, in Brevibacterium flavum, we tested six different types of heterologous promoters P ilvA , P kan , PF104, tac, , tac-M1 and tac-M were tested for activity. P ilvA It is the promoter sequence of threonine dehydrogenase gene ilvA on the chromosome of Corynebacterium glutamicum; P kan is the promoter sequence from transposon Tn5; PF104 is the promoter sequence from Corynebacterium glutamicum phage φGA1; tac is a heterozygous promoter; tac-M1 and tac-M are the tac promoters designed by ourselves derivative.

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Abstract

The invention discloses a corynebacterium promoter detection vector and a construction method and application thereof, and belongs to the technical field of genetic engineering. The corynebacterium promoter detection vector pDXW-11 can be used for screening promoters which can be efficiently expressed in corynebacteria; by detecting plasmids, a strong corynebacterium promoter tac-M which can be efficiently expressed in the corynebacteria is screened; and the corynebacterium promoter detection vector is particularly suitable for the research and production of metabolites of the corynebacteria.

Description

technical field [0001] The invention relates to a promoter detection carrier and its construction method and application, in particular to a coryneform bacterium promoter detection carrier and a strong promoter screened by using the carrier, belonging 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] Since a large amount of knowledge about the physiology, bioch...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12Q1/68C12N15/113C12R1/15
Inventor 王小元徐大庆谭延振李烨
Owner JIANGNAN UNIV
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