|
Brainstorming（Topic design, literature investigation） |
|
Design and synthesis of gene sequences and primers |
| 3.24-3.30 |
Double digest pML104, sgRNA hybridized, T4 ligation |
| 3.31-4.03 |
Transformation and verification of recombination plasmid |
| 4.04-5.09 |
Construction of recombination plasmid pUC57-pADH1-NcEgt1-tCYC1 |
| Construction of recombination plasmid pUC57-H1-pADH1-NcEgt1-tCYC1-H2 |
| 5.10-5.14 |
Transformation with donor DNA and recombination plasmid |
| 5.15-5.21 |
Selection of positive transformants and loss of sgRNA/Cas9 expression plasmid |
| 5.22-5.28 |
Fermentation culture of recombinant strain and detection of target product by HPLC
|
| 5.29-6.03 |
Detection of target product by LC-MS |
| 6.04-6.16 |
Construction of recombination plasmid pTEF2-pTEF1-CpEgt2-tCYC1 |
| Construction of recombination plasmid pTEF2-H1-pTEF1-CpEgt2-tCYC1-H2 |
| 6.17-6.22 |
Construction of recombination pML104 targeting Saccharomyces cerevisiae chromosome
11
|
| 6.23-7.04 |
Transformation with donor DNA and recombination plasmid |
| 7.05-7.10 |
Selection of positive transformants and loss of sgRNA/Cas9 expression plasmid |
| 7.11-7.22 |
Testing of EGT production capacity of two engineered strains |
| 7.23-8.02 |
Determination of growth curve |
