Shuttle plasmid carrier, and construction method and application thereof

A technology of shuttle plasmid and vector, applied in the field of shuttle plasmid vector and its construction, can solve problems such as difficulties in foreign genes

Active Publication Date: 2018-03-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a shuttle plasmid vector to solve the technical problem of difficult introduction of foreign genes in the above-mentioned Paenibacillus polymyxa

Method used

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  • Shuttle plasmid carrier, and construction method and application thereof
  • Shuttle plasmid carrier, and construction method and application thereof
  • Shuttle plasmid carrier, and construction method and application thereof

Examples

Experimental program
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Embodiment 1

[0030] Example 1 Construction of Escherichia coli-Paenibacillus polymyxa shuttle vector pWBUC01

[0031] Zhang Wendi et al. (Zhang Wendi, Ding Yanqin, Yao Liangping et al., Establishment of Paenibacillus polymyxa SC2 gene knockout system [J]. Acta Microbiology Sinica. 2013, 53(12): 1258-1266.) reported that the plasmid of B. subtilis can be found in Replication and expression in P.polymyxa, so the applicability of B.subtilis plasmids pHT43, pWB980, and pNNB194 to P.polymyxa ATCC842 was tried (Table 1), and it was found that only plasmid pWB980 could be replicated and expressed in P.polymyxa ATCC842, and the transformation The efficiency is 8.58±1.43×10 6 (cfu / μg DNA), so pWB980 was selected as the insert.

[0032] Table 1B. Replication and expression of subtilis plasmid in P.polymyxa

[0033]

[0034] pMD19-T Simple Vector is a special vector for high-efficiency cloning PCR (TA Cloning). It eliminates the multiple cloning restriction sites on the pUC19 vector. Therefore, ...

Embodiment 2

[0053] Example 2 Application of Escherichia coli-Paenibacillus polymyxa shuttle plasmid vector pWBUC01

[0054] 1. Escherichia coli-Paenibacillus polymyxa shuttle vector pWBUC01 transformed into Bacillus subtilis WB800

[0055] The shuttle vector pWBUC01 constructed in Example 1 was transformed into Bacillus subtilis WB800, specifically, including the following steps:

[0056] A, preparation of Bacillus subtilis WB800 competent

[0057] 1) Pick a fresh single colony of Bacillus subtilis WB800 and inoculate it in a 5mL LB liquid test tube, and cultivate overnight at 37°C and 200r / min.

[0058] 2), the bacterial solution is inserted into 50mL LB liquid medium containing 0.5mol / L sorbitol according to the inoculum amount of 5% (v / v), cultivated to OD at 37°C and 200r / min 600 When the temperature is about 2, take all the bacterial liquid in ice bath for 30min, centrifuge at 5000r / min, 4°C for 6min, and collect the bacterial cells.

[0059] 3) Resuspend the cells with 10 mL of p...

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Abstract

The invention discloses a shuttle plasmid carrier, and a construction method and application thereof. The shuttle plasmid carrier is formed by linearizing bacillus subtilis plasmid pWB980 and inserting into a carrier pMD19-T Simple Vector. The shuttle carrier plasmid can be copied in escherichia coli and paenibacillus polymyxa. In the paenibacillus polymyxa, the genetic operation system is not yetmature, the conversion steps are tedious and the conversion efficiency is relatively low; and in the escherichia coli, gene cloning operation is very convenient. The plasmid carrier can complete exogenous gene cloning and recombinant plasmid construction in the escherichia coli, related gene function research and genetic breeding work are developed in the paenibacillus polymyxa conveniently, andthe conversion rate can reach 7.83 +/-1.54*10<5>CFU/ug DNA.

Description

(1) Technical field [0001] The invention relates to a shuttle plasmid vector and its construction method and application. (2) Background technology [0002] Paenibacillus polymyxa is a rod-shaped, spore-forming, Gram-positive bacterium that is mostly isolated from plant rhizosphere soil. In recent years, due to its remarkable plant growth-promoting effect and antibacterial effect, it is generally regarded as a kind of plant rhizosphere growth-promoting bacteria. Paenibacillus polymyxa can produce a variety of antagonistic substances that inhibit the growth of bacteria and fungi, such as polymyxin, cyclobacterin, cholipidin, polypeptide mycetin, oryvalerin and fusaricidin, etc. antibiotics, and because some antibiotics are synthesized by non-ribosomal peptide synthetases, this has attracted the attention of more and more researchers. [0003] Plasmid pWB980 is a Bacillus subtilis expression vector constructed by Wu et al. in 1999 based on the transformation of pUB110. This ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/75C12N15/65
CPCC12N15/65C12N15/70C12N15/75
Inventor 孙东昌裘娟萍沈敏佳
Owner ZHEJIANG UNIV OF TECH
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