Summary (June 1 - August 15, 2024)
This experiment focuses on two major systems: the construction and optimization of strains for kaempferol production and GABA production. Both systems involve genetic engineering techniques and optimization of expression conditions to maximize product yield.
System 1: Kaempferol Production Strain Construction and Optimization
1. Objective: To construct and optimize E. coli strains that can produce kaempferol through the expression of the genes CisF3H and CuFLS.
2. Key Steps:
a. Synthesize the CisF3H and CuFLS genes and clone them into the pET23b plasmid.
b. Transform the plasmids into E. coli strains DH5α for cloning and BL21 for expression.
c. Induce kaempferol production using naringenin and analyze its content using a UV spectrophotometer.
d. Optimize production conditions by testing different parameters, including temperature, initial cell density, naringenin concentration, and linker effects.
3. Expected Results: Efficient kaempferol production with optimal conditions identified for temperature, cell density, and naringenin concentration.
System 2: GABA Production Strain Construction and Optimization
1. Objective: To construct and optimize E. coli strains that can produce GABA through the expression of the GadB gene.
2. Key Steps:
a. Synthesize the GadB gene and clone it into the pET23b plasmid.
b. Transform the plasmids into E. coli strains DH5α for cloning and BL21 for expression.
c. Optimize GABA production through enzyme activity tests under different pH conditions.
d. Measure GABA production levels using an assay kit and analyze enzyme activity at various pH levels.
3. Expected Results: Maximum GABA production with an optimal pH range for enzyme activity identified.
Daily Plan Summary Table (June 1 - August 15, 2024)
System 1: Kaempferol Production Strain Construction and Optimization
| Date | Experiment Title | Experiment Objective | Key Steps | Expected Results | Remarks |
| June 1 June 5 | Synthesis and Cloning of CisF3H and CuFLS | Synthesize and clone genes into pET23b | Gene synthesis, cloning into pET23b using restriction sites | Genes successfully cloned into plasmid | Prepare for transformation |
| June 6 June 10 | Transformation of Plasmid | Transform the plasmid into E. coli BL21 | Transform CisF3H and CuFLS into BL21 strains | Transformants confirmed by colony screening | Prepare for plasmid extraction |
| June 11 June 13 | Plasmid Verification | Verify plasmid sequence integrity | Plasmid extraction, sequencing | Plasmid sequences confirmed to be correct | Begin kaempferol production experiments |
| June 14 June 18 | Induction of Kaempferol Production (F3H-B0034-FLS) | Measure kaempferol production from F3H-B0034-FLS strain | Add naringenin to induce production, analyze kaempferol production | Kaempferol production measured | Prepare for constructing the next strain |
| June 19 June 25 | Construction of F3H-GGGS-FLS Strain | Link F3H and FLS with GGGS linker and clone | Clone and transform F3H-GGGS-FLS into BL21, confirm via sequencing | F3H-GGGS-FLS strain successfully constructed | Prepare for kaempferol production measurement |
| June 26 June 30 | Induction of Kaempferol Production (F3H-GGGS-FLS) | Measure kaempferol production from F3H-GGGS-FLS strain | Add naringenin, incubate, and analyze kaempferol production | Kaempferol production measured | Compare production levels with F3H-B0034-FLS |
| July 1 July 5 | Temperature Optimization | Optimize production temperature | Test different temperatures (16°C, 25°C, 30°C, 37°C, 42°C) | Optimal temperature for production identified | Proceed with cell density optimization |
| July 6 July 10 | Cell Density Optimization | Optimize initial cell density | Test different cell densities (OD600 = 0.2, 0.6, 1.0, 1.5, 2.0) | Optimal cell density identified | Proceed with naringenin concentration optimization |
| July 11 July 15 | Naringenin Concentration Optimization | Optimize naringenin concentration | Test naringenin concentrations (125, 250, 500, 1000, 2000 mg/L) | Optimal naringenin concentration determined | Proceed with linker optimization |
| July 16 July 20 | Linker Effects on Kaempferol Production | Study effects of linkers on kaempferol production | Compare flexible (GGGS) and rigid (TPTP) linkers | Rigid linker shows higher production | Proceed with multi-gene optimization |
| July 21 July 25 | Multi-Gene Copy Effects | Test effect of multiple gene copies on production | Analyze the effect of adding multiple copies of F3H and FLS | Multi-gene increases kaempferol yield | Continue optimization trials |
| July 26 July 30 | The mRFP Expression Level Analysis | mRFP Expression Level Analysis in F3H-FLS Recombinant Strains | qRT-PCR | The mRNA expression levels | Prepare for data analysis |
| August 1 August 5 | Final Kaempferol Data Analysis | Analyze final kaempferol production data | Review experimental data | Complete data analysis and prepare final report | Finalize results |
System 2: GABA Production Strain Construction and Optimization
| Date | Experiment Title | Experiment Objective | Key Steps | Expected Results | Remarks |
| July 1 July 5 | Synthesis and Cloning of GadB | Synthesize and clone GadB gene into pET23b | Gene synthesis, cloning into pET23b | GadB gene successfully cloned into plasmid | Prepare for transformation |
| July 6 July 10 | Transformation of Plasmid | Transform GadB plasmid into E. coli DH5α and BL21 | Transformation into DH5α and BL21 strains | Transformants confirmed by colony screening | Prepare for plasmid extraction |
| July 11 July 14 | Plasmid Verification | Verify the plasmid sequence integrity | Plasmid extraction, sequencing | Plasmid sequences confirmed to be correct | Begin GABA production experiments |
| July 16 July 20 | GABA Production Test | Test GABA production using glutamate as a substrate | Incubate with glutamate, measure GABA with assay kit | Initial GABA production observed | Prepare for pH optimization |
| July 21 July25 | pH Optimization | Optimize enzyme activity for GABA production | Test different pH levels (3.6, 4.6, 7.4, 9.2) | Optimal pH for enzyme activity identified | Final GABA Data Analysis |
| August 1
August 5 |
Final GABA Data Analysis | Analyze final GABA production data | Review experimental data | Complete data analysis and prepare for final report | Finalize results |