Establishment of a screening method for high-yielding resveratrol mutants
A technology of resveratrol and mutants, applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as inconvenience
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0028] Example 1. Construction of pTtg and pTtgR plasmids.
[0029] Using DsRed vector (Clontech company, product number 632412) as template, 5'-cacccgcggcagcagtatttacaaacaaccatgaatgtaagtataatccgtgtggaattgtgagcggataa-3'(forward primer, SEQ ID NO: 1) and 5'-ctgccgcgggtgcctaatgagtgagctaac-3'(reverse primer, SEQ ID: 2) were used as template Primers for PCR amplification. PCR system: 0.2 μM each primer, 0.2 mM dNTPs, 10 ng template, 5 μL 10×Pyrobest Buffer, 0.3 μL Pyrobest, make up the volume to 50 μL with deionized water. The PCR reaction conditions were: 94°C for 30s, 50°C for 30s, 72°C for 1min, and the above procedure was repeated for 30 cycles. The resulting PCR product was digested with SacII and then digested with T 4 DNA ligase (purchased from NEB Company) was used for ligation at 4°C for 4 hours, and the ligated product was transformed into Escherichia coli MC1061 (Quanshijin Company) to obtain recombinant bacteria. Extract the plasmid of the recombinant bacteria to ob...
Embodiment 2
[0032] Example 2. ttgR mutant mu3
[0033] Error-prone ttgR gene (SEQ ID NO: 5) was performed with primers 5'-gcacatatggtccgtcgaaccaaagaag-3' (forward primer, SEQ ID NO: 7), 5'-agagagctctcatttgcgcagagccgggc-3' (reverase primer, SEQ ID NO: 8) PCR. The error-prone PCR system is 0.2mM dATP, 1mM dCTP, 1mM dTTP, 0-0.025mMMnCl 2 , 10×PCR Buffer 10μL, rTaq 0.5μL, and deionized water to make up the volume to 100μL. The error-prone PCR reaction conditions are: 94°C for 30s, 50°C for 30s, 72°C for 1min, and the above procedures are repeated for 30 cycles.
[0034] The obtained PCR product was subjected to MEGAWHOPE PCR with pTtgR (SEQ ID NO: 6) as a template, and the reaction system was: 0.2 μg of the PCR product obtained by error-prone PCR above, 1 mM of dNTPs, 30 ng of template pTtgR, 5 μL of 10×PyrobestBuffer, and 0.3 μL of Pyrobest , make up the volume to 50 μL with deionized water. The PCR reaction conditions are: 94°C for 30s, 50°C for 30s, 72°C for 2min, and the above procedu...
Embodiment 3
[0036] Example 3. Construction of plasmid containing pTtgR or pTtgR mu3 recombinant strain BW25113
[0037] Competent BW25113 (Quanshijin) wild-type strain, transformed into pTtgR plasmid or pTtgR respectively mu3 (It is the plasmid obtained in the error-prone PCR process that embodiment 2 screens mutant mu3 to carry out, and in the PCR process, TtgR in the pTtgR plasmid is replaced by mu3), obtains containing plasmid pTtgR or pTtgR mu3 The recombinant strain BW25113.
PUM
| Property | Measurement | Unit |
|---|---|---|
| wavelength | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More