| Date | Experiment | Details | Notes |
|---|---|---|---|
| 6/18 | Gene Sequence Retrieval | Obtain SulE, PnbA, GST sequences from NCBI. | Successfully retrieved sequences. |
| 6/19 | Gene Circuit Design | Draft gene circuits and plasmid maps for each enzyme. | Gene circuits designed; minor issue with overlapping restriction sites, resolved by removing redundant sites. |
| 6/20 | Plasmid Map Finalization | Optimize codons for E. coli; remove EcoRI, XbaI, SpeI, PstI, NdeI, XhoI sites. | Plasmid maps finalized; some codon optimization introduced unintended secondary structure in SulE, fixed manually. |
| 6/21 | Gene Synthesis Order | Order synthesized genes (GST, SulE, PnbA) from Generalbial. | Genes ordered successfully. |
| 6/22 | PCR Amplification Attempt | Amplify synthesized genes for cloning into pET28a(m). | SulE amplification initially failed; optimized annealing temperature and succeeded. |
| 6/23 | Restriction Digestion & Cloning | Digest pET28a(m) and PCR fragments; ligate inserts. | GST and PnbA ligation successful; SulE ligation initially low yield, repeated. |
| 6/24 | Transformation into DH5α | Transform ligated plasmids into DH5α via heat shock (42°C, 1 min). | Transformation successful for GST and PnbA; SulE required second attempt. |
| 6/25 | Screening & Sequencing | Plate on LB + 100 μg/mL kanamycin; pick clones and sequence. | Positive clones obtained; SulE clone had single-base mutation, replaced with correct clone. |
| 6/26 | Plasmid Extraction | Extract plasmids from DH5α clones for BL21 transformation. | Extraction successful. |
| 6/27 | BL21 Transformation | Transform plasmids into BL21 (DE3). | Transformation successful; SulE BL21 grew slowly, repeated induction later. |
| 6/28 | IPTG Induction Trial | Induce expression of enzymes in BL21 strains. | GST and PnbA expressed well; SulE expression weak, increased IPTG concentration. |
| 6/29 | Crude Enzyme Extraction | Lyse cells and collect crude enzyme. | Extraction successful; SulE enzyme partially degraded, adjusted lysis protocol. |
| 6/30 | WB Validation | Verify protein expression via Western blot. | All three enzymes detected; SulE band faint, required longer exposure. |
| 7/1 | Chlorimuron-E Standard Curve | Prepare serial dilutions of chlorimuron-E; ELISA measurement. | Standard curve established; minor pipetting errors in first replicate, repeated. |
| 7/2 | ELISA Replication | Repeat ELISA to ensure accuracy. | Data consistent; curve validated. |
| 7/3 | Enzyme Activity Pre-Test | Test crude enzyme on 20 μg/mL chlorimuron-E for 10 min at 25°C. | PnbA activity high; SulE and GST low; SulE repeat with higher IPTG improved activity. |
| 7/4 | Activity Measurement | ELISA measurement of degraded chlorimuron-E. | Results: PnbA 61.2%, SulE 29.8%, GST 32.3%. |
| 7/5 | Data Analysis | Calculate degradation rates and perform Student t-test (n ≥ 3). | Statistical significance confirmed (p < 0.05). |
| 7/6 | Specific Activity Calculation | Calculate U/μg for each enzyme based on molecular weight and A450 changes. | PnbA: 0.0038; SulE: 0.0019; GST: 0.0021. |
| 7/7 | Temperature Optimization | Test PnbA activity at 25–40°C. | Optimal at 30°C; initial 35°C test caused enzyme denaturation. |
| 7/8 | Temperature Repeat | Repeat temp test around 30°C. | Confirmed 30°C as optimal. |
| 7/9 | pH Optimization | Adjust PBS to pH 5–9; test PnbA activity. | pH 7 optimal; pH <6 caused reduced activity. |
| 7/10 | pH Repeat | Verify pH effect with replicate tests. | Results consistent. |
| 7/11 | INP Surface Display | Clone INP-PnbA fusion into BL21. | Cloning successful; initial surface display low, optimized promoter. |
| 7/12 | INP Expression Validation | Verify INP-PnbA expression on cell surface. | Expression confirmed; some cells lysed, adjusted induction conditions. |
| 7/13 | Whole-Cell Catalysis Trial | React 20 μg/mL chlorimuron-E with BL21-INP-PnbA for 30 min at 25°C. | Partial degradation observed; reaction repeated. |
| 7/14 | ELISA Measurement | Measure remaining chlorimuron-E. | Degraded 28.1 μg/mL; BL21 and BL21-PnbA negligible. |
| 7/15 | Replicate Whole-Cell Test | Confirm degradation efficiency. | Results consistent. |
| 7/16–7/17 | Data Compilation | Compile all degradation, activity, temperature, and pH data. | Data consistent; some outlier in SulE temperature test, removed. |
| 7/18–7/20 | Analysis & Figures | Generate figures for publication/presentation. | Figures completed; some formatting issues corrected. |
| 7/21–7/25 | Final Validation | Repeat key assays for reproducibility; summarize results. | All major findings validated; minor SulE issues noted but did not affect conclusions. |
| Date | Experiment | Method | Notes/Problems |
|---|---|---|---|
| 7/30 | Gene Circuit Design | Draw IAA synthesis gene circuit map | Circuit design completed |
| 7/31 | Gene Sequence Retrieval | Retrieve iaaM and iaaH sequences from NCBI | Sequences successfully obtained |
| 8/1 | Codon Optimization | Optimize codons for E. coli; remove EcoRI, XbaI, SpeI, PstI | IaaH initially had secondary structure issues, resolved after adjustment |
| 8/2 | Cloning into Vector | Clone into pET28a(m) via NdeI/XhoI | IaaM cloned successfully; IaaH failed initially, repeated and succeeded |
| 8/3 | BL21 Transformation | Transform plasmid into E. coli BL21 | Transformation successful |
| 8/4 | Preliminary Expression Induction | Induce with IPTG, extract crude enzyme | IaaH expression weak; IPTG concentration increased |
| 8/5 | WB Verification | Western blot to detect protein expression | Both enzymes expressed; IaaH band faint |
| 8/6 | IAA Standard Curve | Prepare IAA standards and measure A450 via ELISA | Standard curve established; pipetting error required repeat |
| 8/7 | IAA Curve Re-measurement | Repeat ELISA for verification | Data consistent |
| 8/8 | Fermentation Induction | Grow 5 mL LB+kana to OD600=0.6, induce with 0.5 mM IPTG for 12h | Culture grew well; temperature slightly low, some cells grew slowly |
| 8/9 | Supernatant Collection | Centrifuge 1.5 mL culture, measure IAA via ELISA | IAA 14.9 μM; wild-type and single-enzyme strains produced almost no IAA |
| 8/10 | Production Tracking | Measure IAA over 24h | Final IAA 15.09 μM |
| 8/11 | Data Organization | Organize production data | Data consistent |
| 8/12 | Wheat Seed Preparation | Surface sterilize seeds with 75% ethanol 1 min, rinse 3x with sterile water | Seeds sterilized; some floated, gently pressed |
| 8/13 | Seed Soaking | Soak seeds in 0 μM (control) or 10 μM IAA for 4–6h | Soaking uniform; some seeds absorbed insufficiently, extended 30 min |
| 8/14 | Sowing | Place seeds on moist filter paper in Petri dishes, maintain moisture, 25°C | Seeds evenly placed; filter paper slightly dry, added water |
| 8/15 | Germination Observation | Day 1 germination check | Low germination; some seeds didn’t sprout, checked water content |
| 8/16 | Germination Observation | Day 2 recording | Experimental group germination increased; control slower |
| 8/17 | Germination Observation | Day 3 recording | Data normal; some seeds with black spots removed |
| 8/18 | Root & Shoot Measurement | Day 5 measurement | Experimental group height 4.17 cm, root 2.43 cm; control height 3.87 cm, root 2.10 cm |
| 8/19 | Growth Observation | Day 6 recording | Filter paper kept moist; experimental roots slightly curled, monitored water |
| 8/20 | Growth Observation | Day 7 recording | Root elongation significant; experimental group advantage maintained |
| 8/21 | Growth Observation | Day 8 recording | Measure shoot and root length; some control roots beginning to shrink |
| 8/22 | Growth Observation | Day 9 recording | Data recorded normally |
| 8/23 | Root & Shoot Measurement | Day 10 measurement | Experimental height 10.40 cm, root 9.27 cm; control height 8.90 cm, root 5.07 cm |
| 8/24 | Growth Observation | Day 11 recording | Filter paper slightly dry, added water |
| 8/25 | Growth Observation | Day 12 recording | No issues |
| 8/26 | Growth Observation | Day 13 recording | Experimental leaves slightly yellow; checked nutrient solution |
| 8/27 | Growth Observation | Day 14 recording | Data normal |
| 8/28 | Root & Shoot Measurement | Day 15 measurement | Experimental height 17.47 cm, root 15.9 cm; control height 14.83 cm, root 8.23 cm; IAA promoted wheat growth successfully |
| Date | Experiment | Method | Notes/Problems |
|---|---|---|---|
| 9/2 | Low-Temperature Promoter Design | Design gene circuit with PcspA driving mRFP | Circuit design completed |
| 9/3 | Plasmid Construction | Clone mRFP under PcspA into plasmid | Initial ligation failed; repeated successfully |
| 9/4 | BL21 Transformation | Transform plasmid into E. coli BL21 | Transformation successful |
| 9/5 | Preliminary Expression Test | Induce at 16°C and 37°C, observe mRFP fluorescence | Fluorescence strong at 16°C; minimal at 37°C |
| 9/6 | Fluorescence Measurement | Quantify mRFP expression | Data confirmed temperature-dependent expression |
| 9/7 | Self-Destruct Gene Design | Design T4 Holin and T4 Lysozyme gene circuit | Design completed |
| 9/8 | Plasmid Construction | Clone T4 Holin/Lysozyme into plasmid | Initial cloning of Lysozyme failed; repeated and succeeded |
| 9/9 | BL21 Transformation | Transform plasmid into E. coli BL21 | Transformation successful |
| 9/10 | Growth Curve Test at 37°C | Compare wild-type and engineered strain growth | No significant growth difference; self-destruct not induced at 37°C |
| 9/11 | Growth Curve Test at 16°C | Induce self-destruct system | Engineered cells gradually died; system functional |
| 9/12 | Verification | Observe cell death under microscope | Some cells survived longer than expected; increased induction time |
| 9/13 | Data Recording | Record OD600 and cell survival | Data consistent with expected self-destruction |
| 9/14 | Repeat Test | Confirm reproducibility | Repeated; minor variation, confirmed function |
| 9/15 | Summary & Reporting | Summarize low-temp promoter and self-destruct system data | Systems validated; ready for integration with metabolic engineering |
