Rapid high-flux screening technology of beta-glucosidase producing strain
A glucosidase, high-throughput technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low efficiency, high cost, time-consuming, etc., and achieve a simple screening method, less reagent consumption, low cost effect
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Embodiment 1
[0022] Example 1: Rapid screening of active clones producing β-glucosidase from the bovine feces microbial plasmid metagenomic library
[0023] Samples of the plasmid metagenomic library (containing 8,115 single clones) of Miscanthus-fed cow fecal microorganisms were stored in a 96-well plate in a -80°C freezer. Prepare 100 mL of LB medium containing Ampicillin (100 mg / mL) and sterilize it, randomly take some samples from its library and revive 370 single colonies, add 100 μL of Ampicillin (Ampicillin) to each 96-well plate (100 mg / mL) LB medium and 5 μL of preserved bacterial liquid, cultivated at 37 ° C, 120-150 rpm for 10-12 h; the revived bacterial liquid was expanded, and 93 μL of Ampicillin (100 mg / mL) was added to each microwell of a 96-well plate. mL) LB medium and 4 μL of the bacterial solution of the bacteria to be screened, cultured at 37°C, 120-150 rpm for 6-8 hours; for a display reaction, add 3 μL of 10mM pNPG to each microwell of a 96-well plate, and set a negat...
comparative example 1
[0024] Comparative Example 1: Rapid screening of active clones producing β-glucosidase from bovine feces microbial plasmid metagenomic library using aescin plate method
[0025] Prepare 40 mL of LB medium containing Ampicillin (100 mg / mL) and sterilize it, randomly take some samples from its library and revive 370 single colonies, add 100 μL of Ampicillin (Ampicillin) to each 96-well plate (100mg / mL) LB medium and 5μL preserved bacteria solution, cultured at 37°C, 120-150rpm for 10-12h; prepared 3700mL escin medium and sterilized to make 370 culture plates; each took 5μL revived bacteria solution and directly spot On the plate containing escin medium, after inverting at 37°C for 24-48 hours, observe the 370 culture plates that have been inoculated one by one. According to the black hydrolysis circle around the colony, it was preliminarily judged whether the strain produced β-glucosidase. Results A escin plate method was used to screen the library and one positive clone (M16) ...
Embodiment 2
[0026] Example 2: Rapid screening of β-glucosidase-producing functional strains in termite intestinal microorganisms
[0027] Termites were collected in the dead wood forest of Hunan Provincial Botanical Garden, and the intestinal microorganisms of termites were taken in the ultra-clean workbench and dissolved in sterile water, fully shaken and mixed, and then gradiently diluted 10 5 , 10 6 times, and then pipette 0.1mL of the diluted solution onto the LA plate, and culture it at 37°C for 12-24h; 243 strains of bacteria were isolated.
[0028] Prepare 300mL LB medium and sterilize it, inoculate 243 strains of bacteria into 1mL LB liquid medium respectively, culture at 37°C, 180-200rpm for 10-12h for resurrection; Add 93 μL of LB medium and 4 μL of revived bacteria solution, and incubate for 8-12 hours at 37°C, 120-150 rpm; add 3 μL of 10 mM reaction substrate pNPG to a 96-well plate, and react for 40 minutes at 37°C, 120-150 rpm, and set up negative controls and positive con...
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