| Date | Experiment | Details | Notes |
|---|---|---|---|
| 6/29 | Gene Sequence Retrieval & Codon Optimization | Retrieve sequences of AtoB, MvaS, AibAB, LiuC from NCBI. Perform codon optimization for E. coli and remove EcoRI, XbaI, SpeI, PstI sites. | Initial sequences prepared, ready for synthesis. |
| 6/30 | Gene Synthesis Ordering | Submit optimized sequences to Generalbial (China) for synthesis. | Delivery expected in ~5-7 days. |
| 7/1 | PCR Primer Design | Design primers for PCR amplification of synthesized genes; check Tm, GC content, and secondary structures. | Ensure primers compatible with pSB1A3 cloning. |
| 7/2 | Plasmid Backbone Preparation | Linearize pSB1A3 vector; confirm by gel electrophoresis. | Vector ready for cloning. |
| 7/3 | PCR Amplification of Genes | Amplify AtoB, MvaS, AibAB, LiuC fragments from synthesized DNA. | Gel electrophoresis to verify fragment size. |
| 7/4 | PCR Fragment Verification | Run agarose gel; check for correct size and yield. | Some fragments show faint bands, may require re-amplification. |
| 7/5 | Fragment Re-amplification | Re-amplify low-yield fragments; optimize annealing temperature. | Success: clear bands obtained. |
| 7/6 | One-Step Seamless Cloning | Clone PCR fragments into pSB1A3 vector using Seamless Cloning Kit. | Prepare multiple constructs for backup. |
| 7/7 | Transformation into BL21 (DE3) | Transform recombinant plasmids into competent E. coli BL21 cells; plate on LB/Amp. | Colonies appear next day. |
| 7/8 | Colony Screening | Pick single colonies; inoculate in LB/Amp and perform colony PCR to confirm insert presence. | Some colonies fail to amplify; pick additional colonies. |
| 7/9 | Colony PCR Verification | Confirm positive colonies; prepare glycerol stocks for confirmed clones. | Positive clones saved; some clones fail, backup clones maintained. |
| 7/10 | Pre-culture for HMB Production | Inoculate confirmed clones into 5 mL LB/Amp; grow to OD₆₀₀ ~0.6–0.8. | Cultures ready for HMB assay. |
| 7/11 | HPLC Method Setup | Determine λmax for HMB by full-wavelength scan (200–400 nm). | Detection wavelength determined: 208 nm. |
| 7/12 | HPLC Standard Curve | Prepare HMB standard solutions (0.05–2 mg/L) and measure peaks. | Standard curve linear; r² > 0.99. |
| 7/13 | Small-Scale HMB Fermentation | Inoculate clones into M9 medium; 12 h cultivation at 37°C. | OD and supernatant collected for HMB quantification. |
| 7/14 | HMB Quantification | Measure HMB in supernatants via HPLC. | Yield ~60–65 mg/L. |
| 7/15 | Growth Curve Analysis | Monitor OD₆₀₀ of wild-type vs engineered strains. | No significant growth inhibition observed. |
| 7/16 | Glucose Consumption & HMB Correlation | Measure glucose and HMB every 6 h; perform correlation analysis. | Positive correlation observed. |
| 7/17 | Thioesterase Gene PCR Verification | PCR amplify tesB, YciA, MenI; check sequences on gel. | Some fragments faint; re-amplification required. |
| 7/18 | Re-amplification of Thioesterases | Optimize PCR for low-yield genes. | All fragments verified successfully. |
| 7/19 | Thioesterase Cloning into HMB Strain | Clone tesB, YciA, MenI into HMB-producing plasmids. | Prepare constructs for transformation. |
| 7/20 | Transformation & Colony Screening | Transform into BL21-HMB; select colonies and confirm inserts. | YciA clone successful; tesB low efficiency, repeat next day. |
| 7/21 | Re-transformation of tesB | Retry transformation of tesB construct. | Colonies obtained. |
| 7/22 | Small-Scale Fermentation with Thioesterases | Inoculate tesB, YciA, MenI strains into M9 medium; 12 h cultivation. | OD monitored; no growth defects. |
| 7/23 | HMB Quantification with Thioesterases | Measure HMB via HPLC. | YciA: 185 mg/L; TesB: 148 mg/L; MenI: 101 mg/L. |
| 7/24 | Growth & Glucose Analysis | Compare OD and glucose consumption across thioesterase strains. | All strains show normal growth; YciA shows highest carbon utilization efficiency. |
| 7/25 | Replicate Fermentation | Repeat 12 h fermentation in triplicate to ensure reproducibility. | Results consistent; minor variations in yield. |
| 7/26 | Data Analysis & Graphing | Plot HMB production, growth curves, glucose correlation. | Figures prepared for report. |
| 7/27 | Troubleshooting Low-Yield Strains | Investigate MenI low yield; consider promoter strength or codon usage. | Decide to keep for comparison. |
| 7/28 | Final Verification | Repeat key experiments for confirmation. | Data confirmed reproducible. |
| 7/29 | Summary of HMB Expression | Compile HMB yields, growth, carbon flux, and thioesterase effects. | All data documented. |
| 7/30 | Report & Figures Finalization | Prepare experimental report and figures for HMB Expression Verification. | Ready for submission or next experimental phase. |
| Date | Experiment | Details | Notes |
|---|---|---|---|
| 7/31 | Serotonin gene acquisition and codon optimization | TDC (BBa_25P80OSX) and TPH (BBa_25G48CDM) sequences were obtained from NCBI and codon-optimized for E. coli. Restriction sites EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with RFC#10 standard. | Gene sequences prepared for synthesis. |
| 8/1 | Gene synthesis order | The optimized TDC and TPH genes were submitted to Generalbial (China) for synthesis. | Expected delivery 5–7 days. |
| 8/2 | Primer design | Primers were designed for PCR amplification and cloning. | Tm, GC content, and secondary structure checked. |
| 8/3 | Vector preparation | pET28a(m) vector was prepared and NdeI/XhoI restriction sites verified. | Vector ready for cloning. |
| 8/4 | PCR amplification | TDC and TPH fragments amplified, checked by gel electrophoresis. | Clear bands, sufficient yield. |
| 8/5 | Fragment cloning | TDC and TPH fragments cloned into pET28a(m) using NdeI/XhoI sites. | Multiple construct backups prepared. |
| 8/6 | Transformation into DH5α | Recombinant plasmids transformed into DH5α and plated on LB/Kana plates to select positive colonies. | Some colonies grew slowly; extended incubation needed. |
| 8/7 | Colony screening | Single colonies inoculated into LB/Kana liquid medium and sequenced for verification. | Sequencing confirmed positive clones. |
| 8/8 | Transformation into BL21 | Verified recombinant plasmids transformed into BL21 for expression experiments. | Overnight culture obtained colonies. |
| 8/9 | Small-scale preculture | Positive BL21 strains inoculated into 5 mL LB/Kana, cultured to OD₆₀₀ ~0.6. | Ready for induction. |
| 8/10 | Serotonin induction expression | Inoculated into 20 mL modified M9 medium; at OD₆₀₀ = 0.6, 0.5 mM IPTG added, incubated for 20 h. | Sampling scheduled. |
| 8/11 | Sampling and filtration | 1 mL samples collected every 5 h, filtered through 0.22 μm PES membrane, stored at -20°C. | Samples preserved. |
| 8/12 | Serotonin ELISA measurement | Serotonin concentration measured using 5-HT ELISA kit. | Preliminary titer 1.85 mg/L. |
| 8/13 | Functional verification | Compared serotonin production of wild-type, TDC-only, and TDC+TPH strains. | TDC-only strain produced no detectable serotonin. |
| 8/14 | Data recording and analysis | Serotonin titer over time was recorded and plotted. | Used for yield and expression efficiency analysis. |
| 8/15 | Screening TDC from different sources | Tested TDC genes from rice, Catharanthus, and pig to evaluate effect on serotonin yield. | Catharanthus-derived TDC performed best. |
| 8/16 | Replicate experiments with different TDC | Repeated serotonin production experiment with Catharanthus TDC to verify reproducibility. | Maximum titer reached 2.62 mg/L. |
| 8/17 | Small-scale fermentation repeats | Best TDC strain fermented in 20 mL modified M9 medium three times. | Production stable. |
| 8/18 | OD and growth curve analysis | Compared OD₆₀₀ of different strains to check for growth impact. | All strains grew normally, no significant inhibition. |
| 8/19 | Sample collection and HPLC/ELISA verification | Samples collected again for serotonin measurement to ensure ELISA consistency. | Data consistent. |
| 8/20 | Data analysis and graphing | Plotted serotonin production curves and TDC source comparison graphs. | Figures prepared. |
| 8/21 | Troubleshooting | Checked low-yield or low-expression strains and recorded anomalies. | Some TDC sources performed poorly, kept as controls. |
| 8/22 | Serotonin yield repeat verification | Repeated key experiments to confirm 2.62 mg/L titer. | Results reproducible. |
| 8/23 | System summary | Summarized serotonin synthesis system results, including strain construction, yield, and TDC source comparison. | Data ready for next-stage experiments or reports. |
| 8/24 | Report preparation | Completed serotonin system experimental report and figure compilation. | Prepared for transition to safety system or next project phase. |
| 8/25 | Data review | Final review of experimental data to ensure accuracy and reproducibility. | Experiment completed, data complete. |
| Date | Experiment | Details | Notes |
|---|---|---|---|
| 9/2 | Safety system design | Designed a controllable safety system using arabinose-inducible promoter PBAD (BBa_K808000) to drive expression of E. coli toxin MazF for self-lysis. | Genetic circuit drafted. |
| 9/3 | Promoter test setup | mRFP used as reporter to test arabinose-inducible function of PBAD. | Fluorescence/OD₆₀₀ monitored. |
| 9/4 | Promoter function verification | Tested various arabinose concentrations; Fluorescence/OD₆₀₀ increased with arabinose concentration. | PBAD inducible function confirmed. |
| 9/5 | MazF gene synthesis | E. coli MazF gene codon-optimized to meet RFC#10 standard. | Ready for cloning. |
| 9/6 | Construction of PBAD-MazF cassette | Overlap PCR used to place MazF downstream of PBAD, forming PBAD-MazF expression cassette. | Construct verified by gel electrophoresis. |
| 9/7 | Cloning into plasmid | PBAD-MazF cassette cloned into pSB1A3 via XbaI/SpeI restriction sites. | Plasmid ready for transformation. |
| 9/8 | Transformation into DH5α | Recombinant plasmid transformed into E. coli DH5α; plated on LB/Ampicillin plates. | Positive colonies obtained. |
| 9/9 | Preculture of engineered strain | DH5α-PBAD-MazF and wild-type DH5α inoculated into 5 mL LB + 100 μg/mL Ampicillin, grown overnight at 37°C, 150 rpm. | Activation culture ready. |
| 9/10 | Induction setup | Overnight cultures 1:100 inoculated into 20 mL fresh LB, initial OD₆₀₀ = 0.1; induced at OD₆₀₀ ≈ 0.4 with 2% L-arabinose. | Induction for growth curve assay. |
| 9/11 | Growth curve monitoring | Cell growth monitored using FlexStation 3 microplate reader to generate growth curves. | Observed DH5α-PBAD-MazF growth inhibition under arabinose induction. |
| 9/12 | System effect verification | Compared growth of induced, non-induced, and wild-type controls. | Only induced DH5α-PBAD-MazF showed growth suppression. |
| 9/13 | Data collection and analysis | Collected OD₆₀₀ readings, plotted growth curves. | Verified controllable “suicide switch” function. |
| 9/14 | Repeat induction experiment | Repeated arabinose induction to confirm reproducibility. | System function consistent. |
| 9/15 | Troubleshooting | Checked any unexpected growth in engineered strains; ensured PBAD-MazF properly activated. | Minor adjustments made; system reliable. |
| 9/16 | Summary and report | Summarized results, including strain construction, PBAD inducibility, MazF activation, and growth inhibition. | Safety system verified and ready for integration with metabolic experiments. |
