High-inosine-yield strain breeding method
A strain and inosine technology, applied in the field of biomedicine, can solve the cumbersome and difficult problems of improvement, and achieve the effects of simple and feasible method, improved inosine production ability, and stable genetic expression
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Embodiment 1
[0036] (1) Inoculate the myocardin producing strain (No. CICC21929) purchased from China Industrial Microbial Culture Collection Center into the seed culture solution (100ml containing 2.2g of glucose, 1.4g of yeast extract, 1mL of corn steep liquor, 0.8g of urea, 1.0g of peptone, NaCl 0.25g, Ade 2.5mg, pH 7.0), cultured at 37°C for 24 hours, centrifuged, washed with normal saline for 3 times and resuspended, and configured by plate colony counting method to about 10 8 cells / mL of bacterial suspension. Take 1 mL of bacterial liquid and mix equal volumes of different antibiotic solutions, irradiate with 20W ultraviolet lamp at 30cm for 60s, treat at 37°C and 180rpm for 60min, and immediately dilute with normal saline to terminate the reaction.
[0037] (2) Take 70 μL of the diluted Bacillus subtilis solution and spread it on the plate of LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0) containing sulfaguanidine and antibiotics, 37 Cultivate at ℃ for 24h, t...
Embodiment 2
[0041] (1) Take one loop of activated gentamicin, rifampicin or streptomycin-resistant strains on the plate and insert it into a Erlenmeyer flask containing 25mL of LB liquid medium, shake and culture at 200rpm at 37°C for 20h, and take 1mL The bacterial solution was transferred to another same LB liquid medium, and cultured to the middle and late logarithm under the same conditions.
[0042] (2) Take 10 mL of bacterial liquid in a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Add 1mL SMMP solution: 2×SMM (sucrose 0.5M, maleic acid 0.02M, MgCl 2 ·7H 2 O 0.02M, pH 7.0)+4×PAB (beef extract 1.5g / L, peptone 5g / L, yeast extract 1.5g / L, NaCl 3.5g / L, glucose 1g / L, KH 2 PO41.32g / L, K 2 HPO 4 3.68g / L). Suspend the bacteria, add lysozyme (final concentration: 0.2-1mg / mL), mix well, place in a 37°C water bath, and take samples for microscopic examination at intervals of 15 minutes to observe the formation of protop...
Embodiment 3
[0047] Example 3 High-yield inosine strain ability detection
[0048] (1) The paper chromatography method was used to analyze the ability of the primary screened strain to produce inosine through fermentation. Cut No. 3 filter paper with a length of 30 cm and a width of 20 cm, draw a straight line with a pencil at a distance of 2 cm from the edge of the filter paper, and set a sample point every 2 cm; use a pipette gun to draw 1 mL of fermentation broth into a 1.5 mL small centrifuge tube, and centrifuge at 12000 rpm for 10 min. Take the supernatant. Use a micro-sampler to place the inosine standard or the processed sample to be tested on the corresponding spot on the filter paper, and let it dry naturally or with a hair dryer. Spotting is 5-10 μL each time, and a total of 20 μL is spotted, and the spot diameter is controlled within 4 mm; roll the spotted filter paper into a cylinder, sew it, and place it on a layer filled with a developing agent about 5 mm deep In the analy...
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