Mutagenesis method for improving mutation probability of active streptomycete producing nosiheptide
A technology of active streptomyces and nosiheptide, applied in the direction of mutant preparation, etc., can solve the problems of low energy of low-energy ions, short theoretical range, etc., and achieve the effect of widening mutations, increasing mutation probability, and relieving fatigue effects
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Embodiment 1
[0032] Spore suspension preparation:
[0033] Take a slant produced by active Streptomyces NX-9, add 100mL sterile water to the slant under the ultra-clean bench, scrape off the spores on the slant, transfer it to a 250mL sterilized Erlenmeyer flask, and add an appropriate amount of sterilized water to it. The glass beads were placed on a shaker and shaken for 30 minutes, the spore liquid was broken, and filtered with a funnel containing sterilized filter paper to obtain a single spore suspension of active Streptomyces;
[0034] dilution:
[0035] Dilute the active Streptomyces monospore suspension to 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 , respectively take 0.1mL coated plate culture medium, place it under the conditions of temperature 28-30℃ and humidity 20-60% and cultivate it for 7-8 days, calculate the number of colonies in the plate, the number of colonies in the plate is preferably 100-200 , Gradient 10 -3 and 10 -4 Standards compliant;
[00...
Embodiment 2
[0061] A mutagenesis method for increasing the mutation probability of active streptomyces, the steps are as follows:
[0062] (1) Take a slant produced by active Streptomyces NX-9, add 100mL sterile water to the slant under the ultra-clean bench, scrape off the spores on the slant, transfer it to a 250mL sterilized Erlenmeyer flask, and add an appropriate amount of Place the sterilized glass beads on a shaker and shake for 30 minutes, break up the spore liquid, and filter it with a funnel containing sterile filter paper to obtain a single spore suspension of active Streptomyces;
[0063] (2) Dilute the active Streptomyces bacterial liquid prepared in step (1) to 10-20cfu / mL, and then spread it on a plate medium containing 0.01% nosiheptide by mass percentage, and carry out a gradient dose of low-energy ion beam Injection mutagenesis, the vacuum degree of the target chamber is 8.0×10 -3 Pa, the injection pulse dose is set to 90×2.6×10 13 ions / (cm 2 s), after the injection i...
Embodiment 3
[0076] A method for improving the mutation probability of active streptomyces, the steps are as follows:
[0077] The same steps as in Example 2 were adopted, except that the screening factor was replaced by kanamycin, and NX-9 was still used as the control strain. Example 3 also selected 60 single bacterial colonies, and the preliminary screening results are shown in the table below, and 7 of the 60 single bacterial colonies were significantly higher than the control.
[0078] Table 5 Primary Screening Results
[0079] Strain number
Initial screening potency (U / ml)
Lift rate (%)
NX-9
2905
0
NX9-130
3150
8.43
NX9-139
3450
18.8
NX9-144
3650
25.6
NX9-156
3500
20.5
NX9-166
3690
27.0
NX9-167
3560
22.5
NX9-177
3765
29.6
[0080] Insert 7 single colony slants with higher titers into the seed medium. After the cultivation is completed, inocu...
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