Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof
A technique for gene deletion of Streptomyces clavicularis, applied in the field of bioengineering, can solve the problems of no single crossover integration into chromosome, lat gene deletion mutation, cumbersome steps, etc., and achieve the construction method is simple and easy, with strong purpose and high work intensity small effect
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Embodiment 1
[0036] Construction of recombinant plasmid pKCLHS
[0037] ① Extraction of total DNA of Streptomyces clavulatus
[0038] Pick the solid ISP (ISP (Difco): 0.8%, agar powder: 2%, pH = 7.3) on the plate and cultivate it at 28°C for 3 days in the inoculum of Streptomyces clavulicularis in the liquid ISP medium, culture it on a shaker at 28°C for 2 days, and centrifuge The bacterial cells were harvested, and the bacterial cells were washed once with 10 mM EDTA at pH=8.0 or twice with water. Add SET (10mmol / LTris-HCl(pH8.0), 10mmol / L NaCl, 1mmol / LEDTA(pH8.0)) solution to 1mL, and add 20μL of 50mg / mL (or an equivalent amount of other concentrations of lysozyme), 37°C water bath for 30-60min. Add 20 mg / mL proteinase K 28 μL and mix well, add 120 μL 10% SDS, invert and mix well, and incubate at 55° C. for 2 h. Divide the mixture into two clean centrifuge tubes (0.8mL / tube), add 500μL of phenol / chloroform respectively, shake vigorously and mix thoroughly, and carefully draw the super...
Embodiment 2
[0055] Construction of recombinant plasmid pKCLES
[0056] For the specific method, see ①, ②, ③, and ④ four steps in Example 1, and the ⑤ step is different from the restriction endonuclease used in the ⑤ step in Example 1, as follows:
[0057] The resulting recombinant plasmid pKC1139-lat was subjected to EcoRI-ScaI double enzyme digestion, a section of EcoRI-ScaI gene fragment with a size of about 1.4kb was cut out of the lat gene, and about 7.0kb linear pKCLES fragment was reclaimed (DV805A glue recovery kit of Takara Company), Use T on both sticky ends 4 DNA polymerase (product of Takara Company) was used to make up the level, and the leveling system was as follows: sterilized double-distilled water: 3.5 μL, T4 DNA polymerase buffer (10×): 1 μL, 0.1% BSA: 1 μL, recovered pKCLES: 2.5 μL, dNTP (2mM): 1μL, the total system of filling up reaction: 9μL; the system was first incubated at 70°C for 5min, then transferred to 37°C and added 0.5μL 4U / μL T 4 For DNA polymerase, mix...
Embodiment 3
[0061] Derivative 1 of Escherichia coli-Streptomyces shuttle plasmid containing partial deletion of Streptomyces clavulicularis lat gene and its construction method
[0062] For the specific method, see ①, ②, ③, and ④ four steps in Example 1, the ⑤ step is different from the restriction endonuclease used in the ⑤ step in Example 1, and the specific steps are as follows:
[0063] Use any two enzymes in the remaining restriction sites (see Figure 3) except HinoIII-ScaI and EcoRI-ScaI used for constructing plasmids pKCLHS and pKCLES on the lat gene, such as using EcoNI-HindIII for plasmid pKC1139-lat Cut the lat gene of the lat gene, cut off a section of EcoNI-HindIII gene fragment with a size of about 0.7kb, reclaim about 7.6kb linear fragment, and fill in the two sticky ends with T4 DNA polymerase (product of Takara company). The leveling system is as follows: sterilized double-distilled water: 3.5 μL, T4 DNA polymerase buffer (10×): 1 μL, 0.1% BSA: 1 μL, recovered DNA fragment...
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