Date

Contents

Aug 6-7

1. Prepared liquid and solid LB medium
2. Prepared antibiotic stock solutions
3. Defined PCR system and conditions
4. Made 1% agarose gel
5. Performed inoculation for bacterial liquid culture

Aug 8-12

1. Extracted and purified pET28a and pETDuet-1 plasmids
2. Carried out double digestion with a restriction enzyme
3. Performed agarose gel electrophoresis and gel extraction
4. Performed homologous recombination and transformation (partial failure, requiring re-preparation)

Aug 13-16

1. Observed colony growth for qualitative detection
2. Conducted colony PCR to verify recombinant plasmids
3. Extracted pETDuet-AGE plasmids
4. Conducted enzyme digestion, gel extraction, homologous recombination, and transformation
5. Prepared reagents and systems for PCR

Aug 17-20

1. Observed colony morphology and growth status
2. Conducted colony PCR and gel electrophoresis
3. Checked inoculated colony status (partial failure, requiring repetition)
4. Cultured colonies for protein analysis
5. Prepared SDS-PAGE gel

Aug 21-23

1. Extracted plasmids and sent them for sequencing (re-cultured some samples)
2. Obtained BL21 bacterial pellets
3. Performed ultrasonic cell disruption and collected the supernatant
4. Conducted protein purification
5. Prepared samples and reagents for SDS-PAGE and enzyme activity assay

Aug 24-25

1. Completed SDS-PAGE, followed by staining and destaining
2. Determined AGE enzyme activity and performed thin-layer chromatography (TLC) analysis

Aug 26-27

1. Performed TLC analysis for NAL-catalyzed reactions (failed due to deteriorated running buffer)
2. Prepared materials and conditions for whole-cell biocatalysis
3. Conducted ELISA experiment (control group requiring repetition)

Aug 28-29

1. Expanded bacterial culture and induced protein expression
2. Collected and washed cells
3. Optimized the formulation of the running buffer
4. Analyzed TLC results of samples with different reaction times

Aug 30

1. Clarified task assignments for each team member
2. Designed the academic defense PPT and practiced once

Aug 31

1. Conducted a simulated academic defense to collect feedback for further improvements

Middle September

1. Each team member proceeded with their designated tasks and drafted the wiki content under their responsibility

End of September-Middle October

1. Conduct two rounds of mock defenses to adapt to the process and ensure full preparation for the formal academic defense