A kind of dovemycin biosynthetic gene cluster and its application
A technology of dovemycin and biosynthesis, applied in the fields of plant genetic improvement, application, microorganisms, etc.
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Embodiment 1
[0065] Genomic DNA extraction of dovemyces-producing strain Streptomyces sp.ZJ306:
[0066]Inoculate fresh Streptomyces sp.ZJ306 mycelium into 50mL TSB medium (17g tryptone, 3g plant peptone, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, add water to 1L, pH7.2-7.4), 28-30°C, shaking culture for about 3-4 days, and centrifuge at 4000rpm for 10 minutes to collect mycelium. Wash the mycelia twice with STE solution (NaCl75mM, EDTA25mM, Tris-Cl20mM), add 30mL of STE solution and lysozyme with a final concentration of 3mg / mL to the washed mycelia, vortex evenly, and incubate at 37°C for 3 hours , add proteinase K to a final concentration of 0.1-0.2mg / mL, mix well, incubate at 37°C for 10 minutes, add SDS to a final concentration of 1-2%, mix well, put in a water bath at 55°C for about 1 hour, and invert the number during the period Second-rate. Add an equal volume of phenol-chloroform-isoamyl alcohol (V / V / V=25:24:1), mix well, and place on ice to cool for ...
Embodiment 2
[0068] Construction of whole genome library of dovemyces-producing strain Streptomyces sp.ZJ306:
[0069] First, through a series of dilution experiments to determine the amount of restriction endonuclease Sau3A I, in a 20 μL system, containing 17 μL of genomic DNA, 2 μL of 10 × reaction buffer and 1 μL of different dilutions of Sau3A I, which terminates the reaction 4 μL 0.5mol / L EDTA and a suitable loading buffer. Through exploration, it is determined that the enzyme activity unit of 0.025-0.05U is more appropriate. On this basis, a genomic DNA fragment slightly larger than 40kb was obtained by a large number of partial enzyme digestions, and dephosphorylated with a dephosphorylase.
[0070] The vector SuperCos l plasmid used to construct the library was first cut from the middle of the two cos sequences with restriction endonuclease Xba I, then dephosphorylated, and then used restriction endonuclease Bam from the multiple cloning site HI is cut to obtain two arms. The tr...
Embodiment 3
[0073] The establishment of the genetic transfer system of the dovemycin-producing strain Streptomyces sp.ZJ306 and the acquisition of the gene interruption mutant strain, taking the knockout of the Michael-like cyclization reaction enzyme gene ikaC as an example:
[0074] In vitro knockout mutants were obtained by PCR-targeting method. According to the obtained dovemycin biosynthetic gene cluster sequence, a pair of ikaC gene knockout primers were designed referring to the PCR-targeting system reported in the literature. The primer sequences are shown in the ikaC knockout primers ikaCKF / ikaCKR in Table 2. Then refer to the PCR-targeting method to construct the in vitro knockout plasmid and then transfer it into the conjugatively transferred donor bacteria. The specific steps are as follows: (1) transform the cosmid plasmid pCSG3500 into E. coli E.coliBW25113 / pIJ790 to obtain E.coli BW25113 / pIJ790 / pCSG3500, induce the expression of the λ / red recombinant system with 10 mmol / L o...
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