Multi-copy beefy meaty peptide expression gene and carrier, and recombination Pichia pastoris construction
A technology for expressing genes and beef flavor, which is applied in the construction of multi-copy beef flavor peptide expression genes and vectors and recombinant Pichia pastoris, which can solve the problem of excessive linking reagents and acyl carriers, linking reagents affecting products and the environment, and limiting protein hydrolysates Use and other issues to achieve the effect of optimizing selectivity, reducing difficulty, and increasing output
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Examples
Embodiment 1
[0033] Example 1: Construction of 16 copies of BMP
[0034] The self-designed plasmid vector containing 4 copies of BMP expression gene (synthesized by Dalian Bao Biological Company) was digested with Xho I and Sal I, and the digestion system was subjected to agarose gel electrophoresis, and the 106bp size was recovered Small snippet. Then the designed plasmid vector containing 4 copies of BMP expression gene was single-cut with Xho I, and then dephosphorylated with alkaline phosphatase to prevent self-ligation of the plasmid vector, and then the recovered 106bp small fragment was ligated with the linearized vector . The ligation product was transformed into competent E. coli by electroporation, and the bacterial solution was spread on an LB solid plate containing ampicillin. After 16 hours incubation at 37°C, pick the positive transformants that grow on the plate, extract the plasmids, use Xho I and Sal I double enzyme digestion to verify, select transformants that can cut out 20...
Embodiment 2
[0035] Example 2: Construction of expression vector pPIC9
[0036] The selected transformant plasmid into which 16 copies of the BMP expression gene are inserted forward is then digested with EcoR I and Not I, and the cut small fragments are recovered for use. The expression vector pPIC9 was double digested with EcoR I and Not I and then ligated with the above small fragments. The ligated product was transformed into competent E. coli by electroporation through the ligated product, and the bacterial solution was spread on the LB solid plate containing ampicillin . After culturing at 37°C for 16 hours, the positive transformants growing on the plate were picked, and the plasmid was extracted and verified by PCR.
Embodiment 3
[0037] Example 3: Construction of Pichia pastoris engineering strain
[0038]The verified positive transformant plasmid (pPIC9 containing 16 copies of the BMP gene) was digested with Sac I, linearized, and electrotransformed into Pichia pastoris GS115, and the bacterial solution was spread on the MD plate. After culturing at 28°C for 50 hours, the positive transformants were picked, and the host DNA was extracted for PCR verification.
PUM
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com