Serial duoble function kes carrier suitable for streptomycete chromosome gene knock-out

A technology of chromosome gene and streptomyces, which is applied in the field of gene carrier to achieve the effect of convenient screening

Inactive Publication Date: 2005-03-02
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After reviewing the literature of the prior art (Kieser T, Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A.: Practical Streptomyces genetics "Streptomyces Genetics Operating Manual". Norwich, England: The John Innes Foundation; 2000 ) search found that using this vector can certainly construct the Streptomyces gene library in Escherichia coli and facilitate the structural analysis of the cloned fragments, but it is often necessary to return the cloned DNA fragments to Streptomyces for functional analysis. Encountered many obstacles, such as the preparation and regeneration of protoplasts, the host's restriction on the existence of foreign DNA, etc.

Method used

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  • Serial duoble function kes carrier suitable for streptomycete chromosome gene knock-out
  • Serial duoble function kes carrier suitable for streptomycete chromosome gene knock-out
  • Serial duoble function kes carrier suitable for streptomycete chromosome gene knock-out

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of pJTU194 and pJTU413 gene knockout constructs of Streptomyces lividans 1326 chromosome

[0053] Two DNA fragments of 3830bp and 4497bp in the same direction on both sides of the dnd gene cluster (6462bp) on the chromosome of Streptomyces lividans 1326 were cloned into the HpaI sites of pJTU408 and pJTU412, respectively, to construct the gene knockout structure pJTU194 and pJTU413. See the build process figure 2 , P represents the PvuII restriction site.

Embodiment 2

[0055] Intergeneric conjugative transfer of gene knockout constructs pJTU194 and pJTU413 from Escherichia coli to Streptomyces

[0056] In 2ml of LB liquid medium (tryptone 10g, yeast extract 5g, NaCl 5g, distilled water Inoculate Escherichia coli ET12567 (pUZ8002+pJTU194) or ET12567 (pUZ8002+pJTU413) in 1000ml, pH7.0), and inoculate 5ml of LB liquid culture containing the same concentration of antibiotics at a ratio of 1:10 after 12hrs at 37°C culture medium at 37°C with rotation (220rpm) for 2.5hrs and then washed twice with LB liquid medium. Suspend the spores of Streptomyces lividans 1326 as the acceptor in 5ml of 0.05mol / L TES buffer at pH 8.0, heat shock in a water bath at 50°C for 10min, cool with tap water and add an equal volume of spore pre-germination medium (Difco Yeast extract 1g, Difco casamino acids 1g, CaCl 2 0.01M (need to prepare 5M stock solution, add to yeast extract / casein amino acid solution after separate sterilization), distilled water 100ml), 37°C r...

Embodiment 3

[0058] Screening of knockout strains

[0059] The acquired thiostrepton resistance (Thio R ) conjugative transferons, after 7 days of relaxation culture on the non-resistant SFM plate, copied to the SFM plate containing thiostrepton (10 μg / ml), and the screening phenotype was thiostrepton sensitive (Thiostrepton S ) strains, these strains may be gene knockout mutants.

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Abstract

The serial double function cos carriers, including pJTU390, pJTU391, pJTU407, pJTU408, pJTU409, pJTU410, pJTU411 and pJTU412, suitable for streptomycede chromosome gene knock-out feature that each of them consists of following DNA elements: colibacillus plasmid Coie1 duplicating initiation site ori, ampicilin resistance gene bla, streptomycete plasmid pIJ101 duplicating initiation site and duplicon, thiactin resistance gene tsr, lambda bacteriophage cos site, RP4 conjugal transfer start site oriT, promoter capable of being identified specifically by T3 and T7 bacteriophage RNA polymerase, and neomycin resistance gene neo from Tn5 and containing no promoter. The present invention contains colibacillus plasmid, streptomycete plasmid duplicon and resistance screening mark simultaneously, has functions of autonomous duplicating and inheriting simultaneously in two kinds of hosts, and facilitates in vitro genetic operation and gene function analysis.

Description

technical field [0001] The invention relates to a gene carrier in the technical field of biological genes, in particular to a series of bifunctional cos carrier suitable for gene knockout of Streptomyces chromosome. Background technique [0002] Streptomyces, a group of aerobic Gram-positive bacteria with branched filaments, are one of the main microbial groups in soil. Compared with other biological groups, one of the biggest characteristics of its genome is that its DNA has a very high G+Cmol%, as high as 69-78%, which is one of the biological groups with the highest G+Cmol% content known so far. Although Streptomyces belongs to prokaryotes, it has an extremely complex cell differentiation mechanism, and it is a good model material for studying the regulation mechanism of gene expression in time, space and program. Secondly, Streptomyces is one of the biological groups with the most commercial application value in industrial microorganisms. Nearly 70% of the antibiotics i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76
Inventor 邓子新孙宇辉周秀芬贺新义
Owner SHANGHAI JIAO TONG UNIV
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