Overview
In order to correctly understand the daily wet-lab progress of our team and analyze the experimental results timely, we record the experimental contents for every experiment, and finally organize them. The compiled notebook is as follows.
- Preliminary investigation of all three modules in our project.
- Find feasible protocols for wet lab experiments.
Project Determination
- Decide the framework and technical route of our projects.
- Scheme the upcoming wet lab experiments.
Pathway Design and Module Viability Assurance:
- Conduct an in-depth analysis of the chosen pathway to ensure its efficiency and viability.
- Evaluate the compatibility of individual modules within the pathway.
Plasmid Design and Codon Optimization:
- Optimize codons to enhance protein expression levels.
- Prioritize plasmid designs that facilitate downstream processes.
Plasmid Processing and Transformation:
- Place an order for plasmids from Genscript.
- Employ heat shock transformation in E. coli. -Conduct monoclonal screening and extend culture for transformed E. coli.
Primer Design and PCR Verification:
- Design primers for targeted gene sequences with high specificity.
- Order primers from a reliable supplier. -Perform PCR and agarose gel electrophoresis verification to confirm the correct transformation of the target gene.
Plasmid Extraction and Standard Curve Construction:
- Extract Opener Top10 plasmid for further experiments.
- Construct a standard curve for GFP protein concentration, aligning it with Relative Fluorescence Units (RFU).
- Initiate preliminary quorum sensing experiments.
Monoclonal Screening and Microplate Reader Calibration:
- Repeat monoclonal screening and extend incubation for successfully transformed BL21(DE3).
- Test and optimize the measurement conditions of the microplate reader for accurate readings.
Plasmid Verification and QS Plasmid Extraction:
- Verify successful plasmid transformation through PCR and Agarose gel electrophoresis.
- Extract QS plasmid from BL21(DE3)
- Incubation of BL21(DE3) (QS contained)
Plasmid Correction and Target Gene Construction:
- Improve some fragments in the plasmid design.
- Construct target genes (PobR and TcpH) using precise primers and PCR techniques.
Transformation, Induction, and Fluorescence Testing:
- Transform the improved plasmid and perform monoclonal screening.
- Induce gene expression and conduct fluorescence tests for QS characterization.
Threshold Guard Switch Modification and Inducer Gradient Concentration Test:
- Modify the Threshold Guard Switch.
- Conduct inducer gradient concentration tests and find the threshold level.
Plasmid Reconstruction and Gel Electrophoresis:
- Reconstruct plasmids using primers and PCR for improved performance.
- Verify the reconstructed plasmids using agarose gel electrophoresis.
Transformation, Monoclonal Screening, and Expression Induction:
- Transform reconstructed plasmids and perform monoclonal screening.
- Induce gene expression and extend incubation for optimal protein production.
Fluorescence Testing for PobR, Nanoluc, and Threshold Guard Switch:
- Perform comprehensive fluorescence tests to evaluate the expression levels of PobR, Nanoluc, and the modified Threshold Guard Switch.
Nanoluc Plasmid Extraction and IPTG Induction Measurement:
- Extract Nanoluc plasmid for downstream experiments.
- Measure the effectiveness of IPTG induction for controlled gene expression.
Protein Purification and SDS-PAGE:
- Purify the proteins expressed in PobR, QS, and QNS experiments for further analysis.
- Determine and identify protein concentrations using SDS-PAGE.
Data analysis and Visualization
- Accomplish the analysis of the data from previous experiments and visualize the results by graphic.
- Photograph the fluorescence expression of the Threshold Guard Switch and Quorum Sensing.
For more detailed notebook description, please download the PDF of our lab record on benchling.
