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
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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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