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Colibacillus-corynebacterium shuttle constitutive expression carrier and construction method thereof

A technology of constitutive expression and coryneform bacteria, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of limited application, low expression level, low transcriptional activity, etc.

Active Publication Date: 2013-05-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of these vectors have two disadvantages: one is that there are fewer single restriction enzyme sites at the multiple cloning site, and the other is that the gene is in the lacI q Under the control of induction expression, and the expression level is low
In addition, in coryneform bacteria, the transcriptional activity of the tac promoter is low (Liebl et al., 1992; Billman-Jacobe et al., 1995; Salim et al., 1997; Xu et al., 2009), resulting in the expression of foreign genes inserted into the vector low, which limits the production of target metabolites in engineered bacteria
In addition, most of the expression vectors in the existing coryneform bacteria are inducible expression vectors, and the inducer IPTG needs to be added in the production process, and IPTG is quite expensive and is not suitable for being used as a gene expression inducer for large-scale production of target products; lactose can Used to replace IPTG as an inducer for large-scale production, and for the vast majority of coryneform bacteria strains, lactose cannot enter their cells (Brabetz et al., 1991), which also limits the inducible expression system in the coryneform Application in Bacillus

Method used

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  • Colibacillus-corynebacterium shuttle constitutive expression carrier and construction method thereof
  • Colibacillus-corynebacterium shuttle constitutive expression carrier and construction method thereof
  • Colibacillus-corynebacterium shuttle constitutive expression carrier and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of expression vector pDXW-10

[0034] The construction process of expression vector pDXW-10 is as follows: figure 1 , As shown in 2.

[0035] First, the artificially synthesized 74bp multiple cloning site L-MCS DNA fragment 5'gaattcgcta gcgagctccc atgggcggcc gcctcgaggg taccagatct ccgcggctta agctgcagaa gctt 3', it contains 11 single restriction endonuclease cut points: EcoRI, NheI, SacI, NcoI, NotI, XhoI, BglII, SacII, AflII, PstI and HindIII; Double cut the 74bp MCS fragment with EcoRI and HindIII, and ligate it to pDXW-6, which is also double cut with EcoRI and HindIII, instead of pDXW- 6 The original multiple cloning site, the L-MCS of the constructed plasmid and its two wings were sequenced to verify that the MCS was correctly connected to pDXW-6. The constructed plasmid was 8351bp in size and named pDXW-9 ( figure 1 );

[0036] Then, by overlapping PCR technology, the tac promoter on pDXW-9 was subjected to site-directed mutation ( figure 2 ). O...

Embodiment 2

[0044] Example 2 Evaluation of the applicability of plasmid pDXW-10 in Brevibacterium flavum

[0045] In order to prove the availability of pDXW-10, we expressed the cat gene in Brevibacterium flavum. Using the commercial plasmid pACYC184 as a template, and primers cat-F and cat-R with SD sequences as primers, the open reading frame of 660bp vhb gene was amplified by PCR, and restrictions were introduced at the 5′ and 3′ ends of the amplified product Enzymes EcoRI and HindIII restriction sites. The PCR amplification reaction system is 50μl, including 10μl 5×PrimerSTAR buffer(Mg 2+ plus), 4μl dNTP mix (2.5mM each), 1μl plasmid template pACYC184 (100ng / μl), 1μl forward primer cat-F (20μM), 1μl reverse primer cat-R (20μM), 0.5μl PrimeSTAR TM HS DNA Polymerase; reaction program: 94°C pre-denaturation minutes; 94°C denaturation for 30 seconds, 54°C annealing for 15 seconds, 72°C extension for 45 seconds, 35 cycles; 72°C for another 10 minutes; storage at 10°C. The PCR product was d...

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Abstract

The invention relates to a colibacillus-corynebacterium shuttle constitutive expression carrier and a construction method thereof, which belong to the technical field of gene engineering. In the invention, the expression carrier pDXW-10 can be reproduced in colibacillus and corynebacterium, exists in the colibacillus and the corynebacterium stably and contains a tac-M promoter with a transcription initiation function and a terminator rrnBT1T2 with a transcription termination function in both the colibacillus and the corynebacterium; the expression carrier pDXW-10 contains a resistance gene for marking resistance marking kanamycin used for selecting a corynebacterium transformant; and the expression carrier pDXW-10 contains multiple cloning sites L-MCS containing 11 single restriction enzyme tangent points. In the corynebacterium, the carrier has moderate level for expressing foreign proteins and is especially suitable for the research of the metabolic engineering of the corynebacterium.

Description

Technical field [0001] An Escherichia coli-corynebacterium shuttle constitutive expression vector and a construction method thereof belong to the field of microbial genetic engineering. Background technique [0002] Corynebacterium is a type of Gram-positive bacteria, belonging to actinomycetes with moderate to high GC content. Since the first isolation of Corynebacterium glutamicum to produce L-glutamic acid (Kinoshita et al., 1957), the three main representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum have been It is widely used in the production of amino acids. In particular, it is pointed out that through DNA-DNA hybridization experiments, there are only small differences between these three different species (Liebl et al., 1991). [0003] As a large amount of knowledge about the physiology, biochemistry and genetics of Corynebacterium glutamicum has been accumulated (Eggeling and Bott, 2005; Burkovski, 2008), ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/77
Inventor 王小元徐大庆谭延振李烨
Owner JIANGNAN UNIV
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