Gene blocking mutant for streptomyces coeruleorubidus and preparation method thereof
A technology of Streptomyces erythraea and Streptomyces erythraea is applied in the biological field to achieve the effects of increasing yield and improving fermentation units
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Embodiment 1
[0024] Embodiment 1dauW partial gene fragment amplification
[0025] Strain: Streptomyces coeruleorubidus SIPI-1482, which produces daunorubicin, can be obtained from Shanghai Pharmaceutical Industry Research Institute.
[0026] Medium: YMB liquid medium, YEME liquid medium (Experimental Manual for Genetic Manipulation of Streptomyces hopwood [M], first edition, Changsha: Hunan Science and Technology Press, 1988).
[0027] Main solutions and buffers: TSE, TEL (Experimental Manual of Genetic Manipulation of Streptomyces Hopwood [M], first edition, Changsha: Hunan Science and Technology Press, 1988), saturated phenol / chloroform.
[0028] method:
[0029] Genomic DNA Extraction of Streptomyces coelicolor
[0030] Inoculate fresh slant spores into a 250ml Erlenmeyer flask filled with 20ml of YMB liquid medium, and culture on a shaking table at 30°C and 230r / min for about 48h. The above-mentioned liquid seeds were inserted into a 250ml Erlenmeyer flask filled with 20ml of YEME l...
Embodiment 2da
[0036] The construction of embodiment 2dauW blocking plasmid
[0037] Strain: Escherichia coli DH5α, purchased from Shanghai Sangon Biotechnology Co., Ltd.
[0038]Plasmid: pKC1139, Escherichia coli-Streptomyces shuttle plasmid, apramycin resistance has a selective effect on both Escherichia coli and Streptomyces, Streptomyces replicons are temperature-sensitive, and cannot replicate autonomously when the temperature is higher than 34 °C (references Bierman M, Logan R, O'Brien K, Seno ET, Nagaraja Rao R, Schoner BE, Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116:43-49).
[0039] Main solutions and buffers: Solution I, Solution II, Solution III (Sambrook J, Fritsch EF, Maniartis T. Molecular Cloning Experiment Guide [M], Second Edition, Beijing: Science Press, 1992.).
[0040] method:
[0041] The PCR fragment amplified above was recovered using the GeneClean II kit from BIO101 Company. Place the DNA elect...
Embodiment 3
[0050] Example 3 dauW blocking plasmid conjugative transfer to Streptomyces coelicolor SIPI-1482
[0051] Strains: Escherichia coli ET12567 (available from ATCC, Cat. No. BAA-525); Streptomyces coelicolor SIPI-1482.
[0052] Plasmid: pYG770.
[0053] Medium: MS (Kieser T, Bibb M. Practical Streptomyces Genetics [J]. Norwich: The John Innes Foundation, 2000.).
[0054] method:
[0055] The correct pYG770 plasmid verified above was transformed into Escherichia coli ET12567, and the transformant ET12567 / pYG770 was obtained by screening on the LB+Apr plate. The transformant ET 12567 / pYG770 was inoculated in 2 ml of LB liquid medium (containing chloramphenicol 34 μg / ml and kanamycin 25 μg / ml), and cultured overnight at 37° C. with shaking. Inoculate 20ml of fresh LB (containing 34 μg / ml chloramphenicol and 25 μg / ml kanamycin) at a ratio of 1:100, and culture until the OD value is preferably 0.4-0.6. Centrifuge with a 50ml centrifuge tube to remove the supernatant (Hitachi CR21G...
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