Screening method for breeding Streptomyces roseosporus strains producing Daptomycin and obtained strains
A technology of Streptomyces roseospora and daptomycin, which is applied in the field of strain breeding, can solve the problems of heavy screening of target strains, non-directional mutation, affecting the efficiency of mutagenesis breeding, etc., and achieves faster strain breeding. The effect of selection process, reduction of production cost, and great promotion value
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Embodiment 1
[0020] Example 1 Streptomycin Screening
[0021]Wild-type Streptomyces roseosporus NRRL11379 obtained from the Culture Collection of the United States Department of Agriculture and preserved in cryopreservation tubes was spread on Gaoshi No. 1 slant and cultured at 28°C for 5-6 days. Wash the spores with sterile water, and spread them on Gaoshi No. 1 plates containing streptomycin at various concentrations of 0.5-2mg / L (its composition is: soluble starch 2.0%, NaCl 0.05%, KNO 3 0.1%, K 2 HPO 4 ·3H 2 O 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.001%, agar 2.0%; said percentages are mass volume percentages), cultured at 28°C for 5 days. Then randomly select the single colony grown on the plate and transfer it to the primary seed medium. The composition and mass content of the primary seed medium are: 30g of TSB, 35g of maltodextrin, dissolved in 1L of distilled water, and the initial pH is 7.0. Then shake culture at 30° C. and 200 rpm shaker for 25 hours. Then, the ...
Embodiment 2
[0030] Embodiment 2 daptomycin screening
[0031] The treatment steps of the wild-type Streptomyces roseospore before coating are the same as in Example 1. Then, the wild-type Streptomyces roseospora were coated on Gaoshi No. 1 plates containing daptomycin at various concentrations of 0.5-3 mg / L, and cultured at 28° C. for 5 days. Then the single colony grown on the plate was transferred to primary seed medium. The steps of seed culture and fermentation culture are the same as in Example 1. The detection and calculation of the fermentation unit of daptomycin in the fermentation broth are also the same as in Example 1.
[0032] The results are shown in Table 2. It can be seen from the results in the table that the more daptomycin-tolerant strains obtained from the plate, the higher the fermentation unit of daptomycin. The positive rate of this screening method is 15-25%, while the positive rate of traditional random selection is less than 1%.
[0033] Table 2. Effect of dap...
Embodiment 3
[0036] Example 3 Bacterial inhibition zone screening
[0037] The treatment steps of the wild-type Streptomyces roseospore before coating are the same as in Example 1. Then the wild-type Streptomyces roseospora was spread on Gao's No. 1 plate and cultured at 28°C for 5 days. Then streak the single colony grown on the plate on the plate of Gao Shi No. 1, and culture at 28°C for 5 days. After the bacterial lawn grows evenly on the plate, use a sterile puncher (aperture diameter is about 5mm) to punch holes on the bacterial lawn, and then put the agar circle in the hole on the surface of the coated Staphylococcus aureus bacterial solution. LB tablet. Incubate at 28°C for 1 day, and screen by comparing the diameter of the inhibition zone. Transfer the corresponding strain of the strain with larger inhibition zone diameter to the primary seed medium. The steps of seed culture and fermentation culture are the same as in Example 1. The detection and calculation of the fermentati...
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