Double function kes carrier suitable for streptomycete chromosome gene knock-out

Abstract

The double function cos carrier suitable for streptomycede chromosome gene knock-out features that it 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, strong streptomycete plasmid pIJ101 incompatibility area sti, 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 may be used easily in knocking out streptomycede chromosome gene, contains colibacillus plasmid, streptomycete plasmid duplicon and resistance screening mark simultaneously and has functions of autonomous duplicating and inheriting simultaneously in two kinds of hosts.

Description

technical field

[0001] The invention relates to a gene carrier in the technical field of biological genes, in particular to a bifunctional Coase carrier suitable for knocking out Streptomyces chromosomal genes. Background technique

[0002] Streptomyces is a kind of aerobic Gram-positive bacteria with branched filaments, and it is one of the main microbial groups in soil. Compared with other organisms, 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 a very complex cell differentiation mechanism and is a good model material for studying the regulation mechanism of gene expression in time, space and program. Secondly, Streptomyces is one of the most commercially applicable groups of industrial microorganisms. Nearly 70% of the antibiotics in nature are produced by Streptomyces and...

Examples

Embodiment 1

[0032] Example 1: Construction of the chromosomal gene knockout structure pHZ1553 of Streptomyces nanchang

[0033] First, the cosmid 11A8 (in pHZ1358( figure 1 A DNA fragment of about 37 kb from the chromosome of Streptomyces nanchang was inserted into the BamHI single restriction site of ), and this cosmid was completely digested with BamHI, and the digested product was purified and self-ligated at a low concentration, and transformed into DH5α, Quickly check transformants to pick clones with minimal inserts. The size of the inserted fragment was detected by double digestion with BamHI+EcoRI. The sizes of the two foreign fragments were 4.8kb and 5.8kb respectively, and they were named pHZ1552. pHZ1070 was completely digested with BamHI, and the 1.4kb apramycin resistance gene aac3(IV) was recovered and inserted into the BamHI single restriction site of pHZ1552 as a selection marker for gene knockout, thus completing the target gene knockout structure pHZ1553 build. See th...

Embodiment 2

[0034] Example 2: Intergeneric conjugative transfer of pHZ1553 from E. coli to Streptomyces

[0035] In 2ml LB liquid culture medium containing ampicillin (100μg / ml), apramycin (30μg / ml), kanamycin (10μg / ml), chloramphenicol (25μg / ml) , yeast extract 5g, NaCl 5g, distilled water 1000ml, pH7.0) inoculate Escherichia coli ET12567 (pUZ8002+pHZ1553), 37 ℃ rotary culture (220rpm) 12hr, inoculate to 5ml containing the same concentration of antibiotics according to the ratio of 1:10 In LB liquid medium, rotate culture (220rpm) at 37° C. for 2.5 hr, and then wash twice with LB liquid medium. Suspend the spores of Streptomyces nanchangus as the recipient in 5ml 0.05mol / L TES buffer solution of 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 ste...

Embodiment 3

[0036] Example 3: Screening of gene knockout strains

[0037] The acquired apramycin and thiostrepton resistance (Thio R April R ) conjugative transferons, and then passed through non-resistant GS plate (soluble starch 20g, KNO 3 1g, K 2 HPO 4 0.5g, MgSO 4 ·7H 2 O 0.5g, NaCl 0.5g, FeSO 4 0.01g, 20g of agar, 1000ml of distilled water, pH7.5) After 7 days of relaxation culture, copy to GS plates containing thiostrepton (5μg / ml) and apramycin (10μg / ml) respectively, and screen the phenotype resistant to apramycin and sensitive to thiostrepton (Thiostrepton S April R ) strains, these strains may be gene knockout mutants.

technical field

[0001] The invention relates to a gene carrier in the technical field of biological genes, in particular to a bifunctional Coase carrier suitable for knocking out Streptomyces chromosomal genes. Background technique

[0002] Streptomyces is a kind of aerobic Gram-positive bacteria with branched filaments, and it is one of the main microbial groups in soil. Compared with other organisms, 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 a very complex cell differentiation mechanism and is a good model material for studying the regulation mechanism of gene expression in time, space and program. Secondly, Streptomyces is one of the most commercially applicable groups of industrial microorganisms. Nearly 70% of the antibiotics in nature are produced by Streptomyces and...

Examples

Embodiment 1

[0032] Example 1: Construction of the chromosomal gene knockout structure pHZ1553 of Streptomyces nanchang

[0033] First, the cosmid 11A8 (in pHZ1358( figure 1 A DNA fragment of about 37 kb from the chromosome of Streptomyces nanchang was inserted into the BamHI single restriction site of ), and this cosmid was completely digested with BamHI, and the digested product was purified and self-ligated at a low concentration, and transformed into DH5α, Quickly check transformants to pick clones with minimal inserts. The size of the inserted fragment was detected by double digestion with BamHI+EcoRI. The sizes of the two foreign fragments were 4.8kb and 5.8kb respectively, and they were named pHZ1552. pHZ1070 was completely digested with BamHI, and the 1.4kb apramycin resistance gene aac3(IV) was recovered and inserted into the BamHI single restriction site of pHZ1552 as a selection marker for gene knockout, thus completing the target gene knockout structure pHZ1553 build. See th...

Embodiment 2

[0034] Example 2: Intergeneric conjugative transfer of pHZ1553 from E. coli to Streptomyces

[0035] In 2ml LB liquid culture medium containing ampicillin (100μg / ml), apramycin (30μg / ml), kanamycin (10μg / ml), chloramphenicol (25μg / ml) , yeast extract 5g, NaCl 5g, distilled water 1000ml, pH7.0) inoculate Escherichia coli ET12567 (pUZ8002+pHZ1553), 37 ℃ rotary culture (220rpm) 12hr, inoculate to 5ml containing the same concentration of antibiotics according to the ratio of 1:10 In LB liquid medium, rotate culture (220rpm) at 37° C. for 2.5 hr, and then wash twice with LB liquid medium. Suspend the spores of Streptomyces nanchangus as the recipient in 5ml 0.05mol / L TES buffer solution of 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 ste...

Embodiment 3

[0036] Example 3: Screening of gene knockout strains

[0037] The acquired apramycin and thiostrepton resistance (Thio R April R ) conjugative transferons, and then passed through non-resistant GS plate (soluble starch 20g, KNO 3 1g, K 2 HPO 4 0.5g, MgSO 4 ·7H 2 O 0.5g, NaCl 0.5g, FeSO 4 0.01g, 20g of agar, 1000ml of distilled water, pH7.5) After 7 days of relaxation culture, copy to GS plates containing thiostrepton (5μg / ml) and apramycin (10μg / ml) respectively, and screen the phenotype resistant to apramycin and sensitive to thiostrepton (Thiostrepton S April R ) strains, these strains may be gene knockout mutants.

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