1. Chassis strain screening
2. Genomic DNA extraction
3. Design of analysis vector
4. Construction of analysis vector
5. Electroporation transformation for construction
6. Construction and observation of subcellular localization strain
7. Design of DAS readthrough simulation expression vectors
8. Design of DAK read-through experiment vectors
9. Construction and observation of translational readthrough analysis strains
10. Design of a DAS gene overexpression vector in Pichia pastoris
11. Construction and linearization of the overexpression vector for the DAS gene in Pichia pastoris
12. Construction of P. pastoris Das overexpression strain by means of protoplast transformation method
13. Determination of growth status of P.p Das overexpression strain in methanol medium
14. Design of the expression vector for the yihX gene in Escherichia coli
15. Construction and linearization of the expression vector for the yihX gene in Escherichia coli
16. The construction of the expression strain of Escherichia coli yihX gene by protoplast transformation method
17. Determination of glucose production of Escherichia coli yihX expression strain in methanol medium
18. Design of the pfk gene knockout vector
19. Construction of pfk gene knockout strains
20. Determination of glucose yield in the recombinant strain yihX-pfk in methanol medium