Alpha-cyclodextrin glucosyltransferase gene and application thereof
A glucosyl, cyclodextrin technology, applied in the directions of transferase, application, genetic engineering, etc., can solve the problems of unfavorable industrial production of CGT, complex activation requirements, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0029] Example 1: Cloning of α-cyclodextrin glucosyltransferase gene
[0030] Extract the total genomic DNA of Paenibacillus macerans thalline with proteinase K-phenol extraction method, use this genomic DNA as template, in primer 1 (5'-CCATATGT CCATGG ATTCACCCGATACGAGC-3’) and Primer 2 (5’-CTCGAGA GAATTC GGATTTTGCCAGTCCAC-3’) for PCR amplification, the amount of each component in the PCR reaction system (total volume 50 μL): 10×Taq DNA Polymerase Buffer 5 μL (Mg 2+ ), 5 μL of 10 mM dNTP mixture (dATP, dCTP, dGTP, and dTTP each 2.5 mM), 1 μL each of cloning primer 1 and primer 2 at a concentration of 10 μM, 1 μL of genomic DNA, 0.25 μL of Taq DNA polymerase, and add nucleic acid-free water to 50 μL.
[0031] The PCR instrument of Biorad was used, and the PCR reaction conditions were: pre-denaturation at 94°C for 4min, followed by a temperature cycle of 94°C for 30s, 55°C for 30s, and 72°C for 2min, a total of 35 cycles, and finally an extension at 72°C for 10min, with a term...
Embodiment 2
[0033] Example 2: Acquisition of α-cyclodextrin glucosyltransferase gene
[0034] The total genomic DNA of Paenibacillus macerans JFB05-01 cells was extracted by proteinase K-phenol extraction method, and the genomic DNA was used as a template in primer 3:5'-CAATGTGGGTCCCACaATGGGCCAGCCG-3' and primer 4:5 '-CGGCTGGCCCATtGTGGGACCCACATTG-3') for PCR amplification. The amount of each component in the PCR reaction system (total volume 50 μL): 10×Taq DNA Polymerase Buffer 5 μL (Mg 2+ ), 5 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL of each primer 3 and primer 4 at a concentration of 10 μM, 1 μL of template DNA, 0.25 μL of Taq DNA polymerase, and add nucleic acid-free water to 50 μL.
[0035] The PCR instrument of Biorad was used, and the PCR reaction conditions were: pre-denaturation at 94°C for 4min, then entering into a temperature cycle of 94°C for 30s, 55°C for 30s, and 72°C for 6min, a total of 35 cycles, and finally extending at 72°C for 6min, w...
Embodiment 3
[0072] Example 3: Preparation and Transformation of Recombinant Escherichia coli BL21 / pET-20b(+) / cgt
[0073] According to Example 2, the vector pMD18-T simple / cgt2 that has eliminated the NcoI site in the cgt gene was double digested with NcoI and EcoRI, and the target fragment containing sticky ends was recovered, and then used the same endonuclease and alkaline phosphatase The digested pET-20b(+) plasmid fragment was ligated with T4 DNA ligase, and the ligated product was transformed into E.coliJM109, and the transformed recipient bacteria were spread on the LB solid plate containing 100 μg / mL ampicillin, and cultured overnight at 37°C. A single clone was picked and cultured overnight in LB medium containing 100 μg / mL ampicillin, the plasmid was extracted and identified by restriction electrophoresis and sequencing, and finally the expression vector pET-20b(+) / cgt containing the correct sequence was obtained, and its E.coli BL21(DE3) was transformed to obtain genetically en...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap