Main Timeline:
The timeline is in monthly increments, with each month normalized to 30 days. Each grid cell corresponds to a three-day interval. The shaded areas within the grid represent time periods designated as workdays.
April：
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Literature Reading |
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Theoretical knowledge learning |
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Project brain storm |
May：
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Literature Reading |
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Project introduction |
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Theoretical knowledge training |
June：
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Theoretical knowledge training |
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Project brain storm |
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Experimental design |
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Laboratory safety training |
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Implement experimental plan |
July:
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Implement experimental plan |
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Project problems discussion |
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Record experiment report |
August:
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Implement experimental plan |
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Project problems discussion |
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Analysis of experimental results |
September:
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Project problems discussion |
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Wiki writing |
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Education |
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Preparation of defense materials |
October:
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Preparation of defense materials |
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Education |
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Project review |
Record of Main Experimental Content
July-August:
1. Inoculate glycerol stock (Escherichia coli K12 containing the target gene fragment and host strain containing the target vector)
2. Plasmid extraction
3. PCR amplification of the target fragment and vector
4. Visualization of the target fragment and vector by agarose gel electrophoresis
5. Gel extraction, separation, and purification of the fragment and vector
6. Homologous recombination to obtain the recombinant vector
7. Transform competent cells
8. Plate plating
9. Verify single clones by colony PCR
10. Inoculate seed stock from a single clone
11. Expand seed stock
12. Plasmid extraction
13. Send plasmid for sequencing
14. Replace the TyrA gene from different sources in plasmid 2 by PCR and homologous recombination
15. First-round fermentation test to select the optimal TyrA gene
16. Replace the RBS by loop PCR
17. Second-round fermentation test to select the optimal RBS sequence
18. Transform plasmid 1 and optimized plasmid 2 into BL21 competent cells
19. Plate plating
20. Verify single clones by colony PCR
21. Inoculate seed culture with a single clone
22. Expand seed culture
23. Induce protein expression with IPTG
24. Sampling during gradient time
25. SDS-PAGE
26. Inoculate LBG medium for large-scale expression
27. Protein expression
28. Purification by nickel column
29. SDS-PAGE
30. Inoculate LBG medium
31. Set inducer concentration and sampling time
32. Detect L-DOPA production using ELISA kit
