This experiment aims to construct and validate a genetically engineered E. coli strain to function as a biosensor for detecting acyl-homoserine lactone (AHL), a key signaling molecule of the Ralstonia solanacearum pathogen. The construction first involves using molecular cloning techniques to assemble components—specifically, the AHL receptor protein QscR and its responsive promoter PQscR from Pseudomonas aeruginosa PAO1, coupled with elements controlling the reporter gene mRFP—into the pSB1A3 vector. The entire project, spanning 21 days, comprises four main stages: construction, verification, functional testing, and data analysis of the engineered strain.
The table below provides a summary of the daily plan, followed by subsequent daily protocols containing detailed steps, results, and chart locations.
| Date | Experiment Title | Key Steps | Experimental Record (Summary) |
|---|---|---|---|
| 2025-06-11 | Engineering Strain Construction Initiation: Cloning Preparation | Sequence verification, restriction digest of vector & insert (XbaI/SpeI), gel purification, ligation reaction setup. | Restriction digest gel electrophoresis showed correct bands; ligation reaction proceeded overnight. |
| 2025-06-12 | Transformation and Plating | Transformation of ligation product into E. coli BL21(DE3) via heat shock, plating on Amp+ LB plates, incubation at 37°C overnight. | Single, isolated colonies appeared on plates. |
| 2025-06-13 | Colony Picking and Pre-culture | Picking 5-10 single colonies, inoculating into LB/Amp+ liquid medium, culturing to mid-log phase. | Culture turned turbid; OD600 reached 0.6-0.8. |
| 2025-06-14 | Colony PCR Verification and Sequencing Submission | Performing colony PCR using culture as template, identifying positive clones via gel electrophoresis, sending positive clone cultures for sequencing. | PCR gel showed target bands; selected 2 clones for sequencing. |
| 2025-06-15 | AHL Stock Solution Preparation | Weighing 3OC12-HSL, C10-HSL, 3OHC10-HSL, dissolving in DMSO to prepare 10 mM stock solutions. | All AHL stock solutions were clear; aliquoted and stored at -20°C. |
| 2025-06-16 | Waiting and Preparation | Awaiting sequencing results; preparing media, antibiotics, and utensils needed for testing. | All testing materials prepared and ready. |
| 2025-06-17 | Sequencing Verification and Glycerol Stock Preparation | Analyzing sequencing results, confirming correct clone, performing larger-scale culture, and preparing glycerol stock for cryopreservation. | Sequencing alignment was correct; successfully prepared engineering strain glycerol stock. |
| 2025-06-18 | AHL Biosensor Test: Single Concentration Activation | Activating engineering strain, treating with 10 µM different AHLs for 3 hours, measuring fluorescence and OD600. | Engineering strain showed clear fluorescent response to 3OC12-HSL. |
| 2025-06-19 | Dose-Response Test: Low Concentration Range | Testing 0.001 µM, 0.01 µM, 0.1 µM 3OC12-HSL, measuring fluorescence and OD600. | Observed trend of increasing fluorescence signal with concentration. |
| 2025-06-20 | Dose-Response Test: High Concentration Range | Testing 1 µM, 10 µM 3OC12-HSL, measuring fluorescence and OD600. | Fluorescence signal saturated at 10 µM. |
| 2025-06-21 | Specificity Test: C10-HSL | Testing 0.001 - 10 µM C10-HSL, measuring fluorescence and OD600. | Engineering strain showed weak or no response to C10-HSL. |
| 2025-06-22 | Specificity Test: 3OHC10-HSL | Testing 0.001 - 10 µM 3OHC10-HSL, measuring fluorescence and OD600. | Engineering strain showed no significant response to 3OHC10-HSL. |
| 2025-06-23 | Data Repeats and Gap Filling | Repeating any anomalous data points to ensure at least 3 biological replicates per data point. | Key data points were well reproduced. |
| 2025-06-24 | Data Compilation and Normalization Calculations | Organizing all raw data, subtracting background, calculating normalized fluorescence values (Fluorescence/OD600). | Obtained a complete, clean normalized dataset. |
| 2025-06-25 | Graph Plotting I: Dose-Response Curve | Plotting dose-response curve for 3OC12-HSL (semi-log plot), calculating approximate EC50 value. | Generated clear dose-response curve. |
| 2025-06-26 | Graph Plotting II: Specificity Comparison Chart | Plotting bar chart comparing normalized fluorescence of the three AHLs at 10 µM. | Generated intuitive specificity comparison chart. |
| 2025-06-27 | Statistical Analysis | Performing significance analysis on key data (e.g., t-test). | Confirmed the response to 3OC12-HSL was statistically significant. |
| 2025-06-28 | Lab Notebook & Report Writing I | Organizing all experimental records; drafting the Materials & Methods and Results sections. | Completed the majority of the initial draft. |
| 2025-06-29 | Lab Notebook & Report Writing II | Analyzing experimental results. | Completed the initial report draft. |
| 2025-06-30 | Lab Notebook & Report Writing III | Completing the Discussion and Conclusion sections; integrating all charts and graphs; performing final proofreading. | Formed a complete draft of the experimental report. |
| 2025-07-01 | Project Summary and Archiving | Final data backup; archiving strains and plasmids; lab cleanup; project summary. | All materials archived; project concluded successfully. |
This experiment aims to construct an engineered E. coli strain capable of producing visible indigo pigment in response to the key signaling molecule of Ralstonia solanacearum - Acyl-Homoserine Lactone (AHL). Based on the previously constructed AHL biosensor, the reporter gene mRFP is replaced with a polycistronic unit for the indigo synthesis pathway (TnaA-B0034-FMO). The entire project runs from July 2 to July 20, 2025, spanning 19 days.
| Date | Experiment Title | Key Steps | Experimental Record (Summary) |
|---|---|---|---|
| 2025-07-02 | Engineering Strain Construction: Cloning Preparation | Sequence verification; XbaI/SpeI double digestion of pSB1A3 vector & synthesized fragment (with TnaA-FMO); Gel purification; Ligation reaction setup. | Digestion successful, ligation reaction established. |
| 2025-07-03 | Transformation and Plating | Heat-shock transformation of ligation products; Plating on Amp+ LB plates; Overnight incubation at 37°C. | Single colonies on experimental plate, no colonies on control plate. |
| 2025-07-04 | Clone Selection and Pre-culture | Picking 12 single colonies, inoculating into LB/Amp+ liquid medium, culturing to OD600=0.6-0.8. | Pre-culture of 12 clones completed, cultures turbid. |
| 2025-07-05 | Colony PCR Verification | PCR using pre-culture as template, agarose gel electrophoresis identification of positive clones. | X/X clones show correct target band. |
| 2025-07-06 | Plasmid Extraction & Sequencing Submission | Plasmid extraction from positive clones; Submission to sequencing company for verification. | Plasmid extraction successful, samples shipped. |
| 2025-07-07 | Sequencing Verification & Glycerol Stock Preparation | Analysis of sequencing results, confirmation of correct clone; Preparation and storage of glycerol stock BL21-HMB at -80°C. | Sequencing error-free, glycerol stock BL21-HMB archived. |
| 2025-07-08 | Indigo Standard Curve Preparation | Preparation of indigo standard solutions at different concentrations; OD620 measurement; Standard curve plotting. | Indigo standard curve established, R² > 0.99. |
| 2025-07-09 | Time-Course Experiment Pilot Run & Medium Optimization | Activation of BL21-HMB; Testing growth and basal indigo production in M9 medium; Optimizing experimental workflow. | Strain grows well in M9 with low background, workflow feasible. |
| 2025-07-10 | Time-Course Experiment: Main Experiment Preparation | Preparation of bulk sterile M9 induction medium, AHL working solutions, and all consumables for multi-timepoint sampling. | All reagents and consumables prepared and ready. |
| 2025-07-11 | Time-Course Experiment: Execution & Sampling (Day 1/Rep1) | Activation and washing of BL21-HMB; Resuspension in M9 induction medium; Addition of 1µM AHLs; Sampling at 0, 2, 4, 6, 8 hours; Centrifugation and supernatant collection. | Completed all timepoint samplings for the first biological replicate. |
| 2025-07-12 | Time-Course Experiment: Execution & Sampling (Day 2/Rep2) | Repeat experiment, performing induction and sampling for the second biological replicate. | Completed all timepoint samplings for the second biological replicate. |
| 2025-07-13 | Time-Course Experiment: Execution & Sampling (Day 3/Rep3) | Repeat experiment, performing induction and sampling for the third biological replicate. | Completed all timepoint samplings for the third biological replicate. |
| 2025-07-14 | Supernatant OD620 Measurement & Data Compilation | Measurement of OD620 for all collected supernatant samples using a plate reader. | OD620 values measured for all samples. |
| 2025-07-15 | Data Analysis I: Kinetic Curve Plotting | Conversion of OD620 data to indigo concentration; Plotting time-course curves of indigo production under induction by three AHLs. | Preliminary kinetic curves generated, showing specific response to 3OC12-HSL. |
| 2025-07-16 | Data Analysis II: Statistical Analysis | Calculation of mean and standard deviation for each group; Performing statistical significance tests (e.g., ANOVA). | Significant difference found between 3OC12-HSL group and controls/other AHL groups. |
| 2025-07-17 | Graph Refinement and Integration | Refining all graphs and integrating them into final report figures. | Final, publication-ready figures generated. |
| 2025-07-18 | Report Drafting I: Introduction & Methods | Writing the project introduction/background and detailed experimental methods section. | Draft of Introduction and Methods sections completed. |
| 2025-07-19 | Report Drafting II: Results & Discussion | Writing the Results section incorporating figures; In-depth analysis and discussion of the results. | Draft of Results and Discussion sections completed. |
| 2025-07-20 | Project Finalization & Archiving | Final review of report; Backing up all data; Archiving strains and experimental records. | Project completed successfully, all materials archived. |
This experiment involved constructing an engineered E. coli strain for enhanced salicylic acid production. The salicylic acid synthesis genes (ICS and IPL) were cloned into a pET28a vector, transformed into E. coli BL21(DE3), and verified to create the BL21-ICS-IPL strain. For production testing, the engineered strain and a control strain were cultured in M9Y medium with IPTG induction. Over 24 hours, samples were collected every 4 hours to measure cell density (OD₆₀₀) and salicylic acid concentration using ELISA. The results demonstrated that the engineered BL21-ICS-IPL strain successfully produced significantly higher levels of salicylic acid compared to the control.
| ate | Experiment Title | Key Steps | Experimental Record (Summary) |
|---|---|---|---|
| 2025-07-24 | Plasmid Construction | Digest, Ligate | Completed double digestion and ligation of vector and insert. |
| 2025-07-25 | Transformation | Heat-shock, Plate | Transformed ligation product into competent cells and plated. |
| 2025-07-26 | Clone Screening | Pick colonies, Colony PCR | Picked single colonies and performed colony PCR screening. |
| 2025-07-27 | Sequencing | Submit samples, Prepare media | Sent positive clones for sequencing. Prepared production media. |
| 2025-07-28 | Awaiting Results | Data organization | Organized data while awaiting sequencing results. |
| 2025-07-29 | Strain Storage | Analyze sequence, Make glycerol stocks | Sequence confirmed. Prepared glycerol stocks of BL21-HMB and control strains. |
| 2025-07-30 | Production - Activation | Overnight culture | Activated seed cultures of BL21-HMB and control strains. |
| 2025-07-31 | Production - Induction/Sampling | Induce, Sample every 4h | Induced with IPTG. Sampled every 4 hours for 24 hours. |
| 2025-08-01 | ELISA Assay | Prepare standard curve, Test samples | Measured salicylic acid concentrations in all samples using ELISA kit. |
| 2025-08-02 | Data Analysis | Calculate concentrations, Plot data | Calculated sample concentrations from standard curve and generated plots. |
| 2025-08-03 | Reporting | Analyze results, Write report | Analyzed data and drafted experimental report. |
| 2025-08-04 to 2025-08-18 | Project Wrap-up | Final review | Conducted final review of report and backed up data. |
