This experiment involves constructing several engineered strains expressing Alkane Monooxygenase (AlkM), BsLac, and INP-BsLac proteins using pET23b plasmid. After plasmid construction and transformation into E. coli DH5α for storage and BL21 for protein expression, the activities of the expressed proteins will be tested. The activity of laccase will be measured using the ABTS assay, and the effect of pH and temperature on laccase activity will be analyzed. The experiment also includes molecular docking to evaluate the binding affinity of ligands to AlkM.
The entire procedure from June 20 can be estimated to take 30 days, covering cloning, transformation, expression, laccase activity analysis, and optimization.
| Date | Experiment Plan | Goal | Notes |
| June 20 | Start cloning AlkM, BsLac, and INP-BsLac into pET23b via NdeI and XhoI | Construct plasmids with synthesized genes (AlkM, BsLac, INP-BsLac) | Use restriction enzymes for cloning, confirm by sequencing |
| June 21-22 | Sequence verification of the recombinant plasmids | Confirm plasmid construction by sequencing | Use Genewiz or Qingke for sequencing |
| June 23-25 | Transform plasmids into E. coli DH5α for storage | Ensure plasmid stability in DH5α for long-term storage | Perform transformation with heat shock |
| June 26-28 | Transform plasmids into E. coli BL21 for protein expression | Prepare engineered strains for AlkM, BsLac, INP-BsLac protein expression | Culture at 37°C with Ampicillin |
| June 29 | Inoculate BL21 strains for expression of AlkM, BsLac, and INP-BsLac | Begin growing BL21 cultures for protein expression | Use LB media with Amp (50 μg/mL) |
| June 30 | Optimize protein expression for AlkM and BsLac | Induce protein expression with IPTG, optimize growth conditions | Sample for Western blot to confirm expression |
| July 1-2 | Analyze protein expression by SDS-PAGE and Western Blot | Confirm the expression of AlkM, BsLac, and INP-BsLac proteins | Analyze molecular weights on SDS-PAGE |
| July 3-5 | Perform molecular docking analysis with AlkM | Evaluate binding affinity of ligands to AlkM using Gnina and visualize with Maestro | Use docking to rank binding affinities |
| July 6-8 | Prepare crude enzyme extracts from BL21 engineered strains | Extract enzymes using ultrasonication and analyze enzyme concentrations | Collect samples for further laccase activity testing |
| July 9-10 | Analyze laccase activity of crude extracts using ABTS assay | Measure laccase activity in BsLac and INP-BsLac strains | Spectrophotometric reading at 420 nm |
| July 11-12 | Test the effect of pH on laccase activity | Measure laccase activity at different pH levels (2.0–6.0) using ABTS as substrate | Use Glycine-HCl and Na2HPO4-citrate buffers |
| July 13-14 | Test the effect of temperature on laccase activity | Measure laccase activity at different temperatures (20–60°C) using ABTS | Maintain constant pH (3.2) during temperature optimization |
| July 15-17 | Optimize reaction conditions for maximum enzyme activity | Determine optimal conditions for pH and temperature | Compare BsLac and INP-BsLac activity profiles |
| July 18-19 | Conduct final laccase activity analysis using spectrophotometry | Final measurements of enzyme activity at 480 nm | Express results as enzyme activity units per dry weight |
| July 20 | Conclude data analysis, prepare reports, and plan follow-up experiments | Summarize findings, create a report, and identify future directions | Compile all experiment notes and results |
The experiment begins on July 1st with the preparation of media and sterilization of equipment, followed by the transformation and screening of Bacillus subtilis and E. coli using plasmid constructs. PCR and plasmid extraction procedures are performed to confirm successful transformations, with sequencing used to verify accuracy.
In the second phase of the experiment, starting from July 16th, protein expression is induced in bacterial strains. The extracted proteins are analyzed using SDS-PAGE to separate them based on size. The experiment continues with Western Blot (WB), transferring the separated proteins to a membrane and detecting them with specific antibodies to ensure correct protein expression.
The experiment concludes on July 20th with the analysis of WB results and the planning of further experiments based on the collected data. The schedule ensures that all necessary steps are taken to validate the success of the plasmid construction and protein expression.
| Date | Experiment Plan | Goal | Notes |
| July 1 | Prepare media and reagents, sterilize equipment | Prepare media and sterilize to ensure a sterile environment | UV sterilization for 30 minutes |
| July 2 | Prepare media for Bacillus subtilis, sterilize equipment | Prepare a contamination-free medium for Bacillus subtilis | Add glucose separately to avoid contamination |
| July 3 | Prepare antibiotic stock solutions (Amp) and LB media | Provide media and antibiotics for transformation experiments | Ensure complete antibiotic dissolution |
| July 4 | Liquid culture of Bacillus subtilis | Start bacterial culture in preparation for transformation | Incubate overnight at 37°C, monitor shaking speed |
| July 5 | Bacillus subtilis transformation and plating | Transform plasmids and screen for positive colonies via plating | Plates should be incubated overnight |
| July 6 | Analyze transformation results, PCR verification of colonies | Verify positive colonies by PCR to confirm successful transformation | Use positive controls to avoid false positives |
| July 7 | Plasmid extraction from positive colonies, measure concentration | Extract plasmids for further experiments | Store extracted plasmids at -20°C |
| July 8 | Repeat PCR for result confirmation | Further verify gene integration in transformed strains | Optimize temperature and primer annealing conditions |
| July 9 | Prepare new media, repeat transformation | Ensure stability of transformation results by repeating experiments | Ensure contamination-free media, appropriate antibiotic concentration |
| July 10 | Amplify target genes via PCR, perform gel extraction | Amplify target gene fragments for cloning and plasmid construction | Ensure high efficiency during gel extraction |
| July 11 | Plasmid construction, Golden Gate assembly | Perform plasmid construction via Golden Gate to integrate target genes | Control enzyme digestion conditions carefully |
| July 12 | Transform plasmids into E. coli, bacterial culture | Transform and culture colonies in preparation for screening | Ensure incubation time is appropriate |
| July 13 | Colony screening, PCR verification | Verify plasmid transformation and integration via PCR | Use high-fidelity polymerase during PCR |
| July 14 | Further plasmid extraction, sequencing | Extract plasmids and send for sequencing to confirm accuracy of construction | Careful analysis of sequencing data to avoid errors |
| July 15 | Analyze experimental results, document process | Confirm plasmid construction success and prepare for subsequent experiments | Record detailed steps, plan for the next phase |
| July 16 | Prepare for protein expression, culture strains | Induce bacterial expression of target proteins, collect samples | Control temperature and inducer concentration accurately |
| July 17 | Protein extraction, start SDS-PAGE | Extract proteins and perform SDS-PAGE to separate proteins by size | Add loading buffer and heat samples before loading |
| July 18 | Complete SDS-PAGE, transfer to membrane for Western Blot | Transfer proteins from SDS-PAGE to PVDF membrane for WB preparation | Ensure proper voltage and timing for membrane transfer |
| July 19 | Perform Western Blot, antibody incubation and detection | Use primary and secondary antibodies for WB to detect protein expression | Ensure accurate incubation time and antibody concentrations |
| July 20 | Analyze WB results, organize data, plan next experiments | Analyze target protein expression, evaluate experiment success | Record detailed analysis and ensure accurate data |
This experiment involves constructing IAA-producing engineered strains via two pathways (IAM and IPA) using synthetic gene sequences inserted into the pET23b plasmid. The plasmids will be transformed into E. coli BL21 for expression. The IAA production in engineered strains will be measured using the Salkowski reagent assay. The supernatants from the strains will then be tested for their effects on seed germination and root growth under normal and petroleum stress conditions.
The entire procedure is expected to take around 20 days, starting on July 20. This timeline includes the cloning of genes, transformation into E. coli, measurement of IAA production, and seed germination/root growth assays.
| Date | Experiment Plan | Goal | Notes |
| July 20 | Clone iaaM, iaaH, aro8, kdc, and puuc genes into pET23b | Construct plasmids for IAM and IPA pathways | Use NdeI and XhoI sites for cloning, verify sequences |
| July 21-22 | Sequence verification and plasmid extraction | Confirm correct insertion of genes into plasmids | Use sequencing and plasmid miniprep |
| July 23 | Transform plasmids into E. coli BL21 for protein expression | Prepare engineered strains for IAA production | Perform heat-shock transformation, plate on LB-Amp plates |
| July 24 | Induce IAA production in BL21 strains | Grow engineered strains and induce IAA production | Incubate at 37°C, 180 rpm, wrap flasks in foil to avoid light degradation |
| July 25 | Measure IAA concentration using Salkowski assay | Detect and quantify IAA in the supernatant | Use Salkowski reagent, read absorbance at 530 nm |
| July 26 | Optimize IAA production conditions | Adjust media composition, induction time, and temperature for maximum IAA production | Perform multiple conditions for comparison |
| July 27-28 | Prepare seeds for germination assay | Treat seeds with supernatant from IAM pathway-engineered strain | Soak seeds for 12 hours, begin germination observation |
| July 29-30 | Record germination rate and growth | Monitor and record the seed germination rate for treated and control groups | Compare treated vs. control groups |
| July 31 | Prepare seeds for root growth assay under petroleum stress | Soak seeds in IAM pathway strain supernatant, transfer to petroleum-stressed soil | Treat experimental group with supernatant |
| August 1-2 | Record and analyze root growth | Monitor root growth of treated and control seeds under petroleum stress | Compare root lengths and general growth patterns |
This experiment involves constructing an alkane-inducible promoter system in E. coli DH5α. The genes AlkS and PAlkB are cloned into the pSB1A3 vector using XbaI and SpeI restriction sites, positioned upstream of the mRFP gene. The transcription factor AlkS is regulated by the constitutive J23100 promoter. The recombinant plasmid will then be transformed into E. coli DH5α, and the promoter will be tested by culturing the recombinant strain in M9 medium supplemented with different concentrations of n-dodecane. The fluorescence output of the mRFP gene will be measured to evaluate promoter activity.
The entire procedure is expected to take 16 days, starting from August 1st. This timeline includes cloning, transformation, testing of the alkane-inducible promoter, and data collection.
| Date | Experiment Plan | Goal | Notes |
| August 1 | Clone AlkS and PAlkB into pSB1A3 | Construct the alkane-inducible promoter in pSB1A3 | Use XbaI and SpeI for cloning |
| August 2-3 | Sequence verification and plasmid extraction | Confirm correct insertion of AlkS and PAlkB into plasmid | Perform plasmid miniprep and sequencing |
| August 4 | Transform recombinant plasmid into E. coli DH5α | Prepare the engineered strain for promoter testing | Heat-shock transformation, plate on LB-Amp plates |
| August 5 | Inoculate and culture engineered strain in LB medium | Activate the strain in LB medium overnight | Incubate at 37°C, 200 rpm overnight |
| August 6 | Transfer to M9 medium with n-dodecane | Induce the alkane-inducible promoter with different n-dodecane concentrations | Include controls with and without alkane |
| August 7 | Measure OD600 and fluorescence of the cultures | Evaluate the mRFP gene expression and promoter response | Use microplate reader to measure fluorescence |
| August 8 | Calculate normalized fluorescence ratio (Fluorescence/OD600) | Analyze the alkane promoter’s induction strength | Compare fluorescence at different alkane concentrations |
| August 9-10 | Repeat induction experiments with varying alkane concentrations | Optimize promoter induction with different n-dodecane levels | Ensure consistency and repeatability |
| August 11 | Test reproducibility of fluorescence measurements | Perform replicates to verify the stability and reproducibility of the system | Measure triplicate values for statistical accuracy |
| August 12-13 | Final data collection and analysis | Summarize the results from fluorescence measurements | Perform statistical analysis |
| August 14 | Prepare final report on alkane-inducible promoter activity | Compile all experimental data, results, and conclusions | Generate graphs and tables for the report |
| August 15-16 | Review and finalize report for submission | Final edits and review of the experimental report | Ensure completeness and accuracy of all sections |
