Thiostreptone biosynthetic gene cluster
A technology for thiostrepton and biosynthesis, which is applied in the field of microbial gene resources and genetic engineering, can solve the problems of complicated operation, high production cost, low yield and the like
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Embodiment 1
[0071]Extraction of total DNA from Streptomyces laurentii ATCC31255, a thiostrepton-producing bacterium:
[0072] 100 μL 1 x 10 8 S.laurentii spore suspension was inoculated into 3mL TSB liquid medium, cultivated at 30°C, 230rpm for about 24hrs and reached the late logarithmic growth phase, then inoculated 2mL into 50mL TSB (containing 10mM magnesium chloride, 0.1% glycine), 30°C, 250rpm After cultivating for about 23 hours, it reached the early stage of the stable growth period, and it was milky yellow and turbid. The mycelium was collected by centrifuging the bacterial solution at 4°C and 3500 rpm for 15 minutes, washed with lysate, and 0.5 mL of pale milky yellow mycelium was collected. Add 10 mL of lysate (containing 5 mg / mL of lysozyme) to 1 mL of mycelium, a total of four tubes, vortex until uniform, and bathe in 37 ° C for 15 min. Add 0.1mL proteinase K (10mg / mL, freshly prepared with lysate), 1mL 10% SDS, mix well and quickly put in 70°C water bath for 15mim, it beco...
Embodiment 2
[0074] Establishment of the genetic transfer system of Streptomyces laurentii ATCC31255, a thiostrepton-producing bacterium:
[0075] Culture E.coli ET12567 containing appropriate plasmids to OD 600 0.3-0.4, the bacterial cells in 30mL LB culture medium were collected by centrifugation, washed twice with an equal volume of LB, resuspended in 2mL LB, and used as E. coli donor cells. Take 500 μL of 20% glycerol spore suspension of Streptomyces laurentii ATCC31255 frozen at -80°C, wash twice with an equal volume of TES buffer (50 mM TES Na, pH 8.0), resuspend in an equal volume of TES buffer, Heat shock at 50°C for 10 min to germinate the spores. Add an equal volume of TSB medium and incubate at 37°C for 2-5hr. Centrifuge and resuspend in 0.5-1mL LB as Streptomyces recipient cells. Mix 100 μL of different concentrations of recipient cells with an equal volume of donor cells and directly spread it on a medium containing 10 mM MgCl 2 After incubating at 30°C for 20 hours, gentl...
Embodiment 3
[0078] Construction of Genomic Library of Streptomyces laurentii ATCC31255, a thiostrepton-producing bacterium:
[0079] Firstly, the total DNA of Streptomyces laurentii ATCC31255 was blown several times with a 1ml syringe, so that the total DNA was randomly broken, and the DNA was subjected to gradient centrifugation using sucrose gradient centrifugation, and the DNA fragments slightly larger than 40kb were collected, dephosphorized, and set aside. pJTU2554 was first cut from the middle of the two cos sequences with EcoRV, and ligated with the prepared 40kb DNA fragment overnight. Thaw PromegaPackagene extract stored at -80°C on ice, immediately add 10ul of the ligation product, flick and mix well, and place at room temperature (about 22°C) for 3hr. Add 445ul Phage buffer, invert to mix; add 25ul chloroform to terminate the reaction, centrifuge to make the chloroform sink to the bottom, and store at 4°C. The strain E.coli LE392 frozen at -80°C was spread on LB medium for rec...
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