Carboxylesterase gene and protein coded by same
A carboxylesterase and gene technology, applied in the field of molecular biology, can solve the problems of few carboxylesterases, no research reports, and few studies on new carboxylesterases
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0018] Using sterile water serial gradient dilution method, the achromobacter was coated on the SBA selective medium, coated 100μl / agar plate (90mm diameter), and the coated plate was inverted and cultured in a 37 °C incubator for 2 days. Pick the colony with a large transparent circle on the SBA selective medium, purify twice on the SBA selective medium, add glycerol with a final concentration of 20%, and store it at -40°C.
[0019] Enzyme activity assay: conversion of p-nitrophenyl ester acetonitrile to p-nitrophenol
[0020] Prepare 10mM p-nitrophenyl ester acetonitrile solution as substrate solution; prepare reaction solution: 1mL substrate solution, 4mL absolute ethanol, 95mL50mM Tris-Hcl (pH8.5). Inoculate the isolated strains in 5mL LB liquid medium, culture at 37°C, shake at 160rpm for 16-18h, centrifuge at 5000g for 10min, take 50μL of the supernatant and add it to 150μL of the above reaction solution, measure the absorbance at 405nm with a microplate reader at 45°C ...
Embodiment 2
[0021] Example 2 PCR amplification, sequence determination and analysis of isolated bacterial strain 16S rRNA gene
[0022] Genomic DNA extraction: Inoculate and isolate the strain obtained in Example 1 in 5 mL of LB liquid medium, culture at 37°C with shaking at 160 rpm for 16-18 h, centrifuge at 12,000 g for 1 min to collect the bacterial cells, and discard the supernatant. Genomic DNA was extracted using the Biospin Bacterial Genomic DNA Extraction Kit (Hangzhou Bioer Technology Co., Ltd.), and the operation was performed according to the kit instructions. The extracted genomic DNA was detected by 0.7% agarose gel electrophoresis, and the DNA concentration was determined by SAM4000 Spectrophotometer. The extracted genomic DNA template was stored at -20°C for later use.
[0023] PCR amplification of 16S rRNA gene: PCR reaction system (total system 25 μL):
[0024] Sterile water 9.5 μL; 2xPremix Ex Taq (Version2.0) 12.5 μL; primer 27F (5 μmol / L, primer sequence 5'-AGAGTTTGA...
Embodiment 3
[0027] Construction of Example 3 Genomic DNA Library and Screening of Positive Clones
[0028] Recovery of small genomic DNA fragments: Genomic DNA spliced by ultrasonic method was subjected to 0.7% agarose gel electrophoresis; about 2-6kb DNA fragments were cut out under ultraviolet light, and the gel weight was calculated; AxyPrepDNA gel recovery kit was used[ Aisigen Biotechnology (Hangzhou) Co., Ltd.], DNA was recovered, and the operation was performed according to the kit instructions.
[0029] Ligation of genomic DNA fragments and vectors: Use T4DNA Polymerase DNA (2-5U / ul) and Klenow Fragment (2-5U / ul) (Dalian Bao Biological Engineering Co., Ltd.) to fill in the ends of the DNA fragments, and operate according to the instructions.
[0030] Ligation reaction system (total system 20 μL): 8 μL sterile water; 2 μL 10×T4 DNA Ligase Buffer; 7 μL DNA; 1 μL carrier DNA; 2 μL T4 DNA Ligase (350 U / μl), react overnight at 16 °C.
[0031] Transformation of recombinant vectors an...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More