Notebook | WHHS-China

The notebooks provide a detailed timeline of a comprehensive iGEM project focused on engineering E. coli strains for glyphosate absorption and degradation. Key activities include the cloning of phnE1/E2 and phnJ genes to enhance glyphosate degradation, along with the construction of a strain expressing phnO for AMPA degradation. In addition, cold-inducible systems were designed, including a PcspA-mRFP reporter for temperature-sensitive fluorescence and a PcspA-mazF suicide system to induce cell death at low temperatures. Throughout the project, key molecular biology techniques—such as gene cloning, PCR, plasmid extraction, transformation, and ELISA assays—were used to measure strain performance, enzyme kinetics, and functionality under different conditions, culminating in the successful creation of engineered strains and detailed experimental results.

Day	Task	Objective	Steps	Expected Results
Day 1	Study Experimental Procedures and Theory	Familiarize students with all experimental steps and principles	- Read related literature and protocols - Discuss the experimental design	Students understand the purpose and methods of the experiment
Day 2	Learn Basic Molecular Biology Techniques	Understand basic techniques such as PCR, plasmid extraction, and transformation	- Train students in PCR, plasmid extraction, and digestion - Watch instructional videos	Students acquire basic experimental skills
Day 3	Prepare Equipment and Materials	Ensure that all equipment and reagents are ready	- Prepare LB media, antibiotics, and PCR reagents - Check lab equipment	Materials and equipment are ready
Day 4	Pilot Experiment	Conduct a small-scale pilot experiment to ensure smooth workflow	- Prepare LB media and test the quality - Perform a small-scale plasmid transformation	Pilot test results are normal, preparing for full experiment
Day 5	Learn Data Analysis Software	Understand how to analyze ELISA and enzyme activity results	- Learn how to use software like GraphPad Prism for data analysis	Students can analyze experimental data

Day	Task	Objective	Steps	Expected Results
Day 6	Synthesize and Optimize phnE1/E2 Genes	Generate codon-optimized phnE1/E2 genes for expression	- Order the synthesized phnE1/E2 genes - Prepare the plasmid vector	Receive synthesized genes and plasmid
Day 7	Clone Genes into pSB1A3 Vector	Complete cloning of phnE1/E2 genes	- Use restriction digestion and ligation to clone the genes into pSB1A3 - Verify via agarose gel electrophoresis	Cloning confirmed with correct size bands
Day 8	Transform Plasmid	Transform the plasmid into E. coli BL21	- Transform recombinant plasmid into E. coli BL21 - Plate on LB + ampicillin plates	Colonies appear on the plate, indicating successful transformation
Day 9	Colony PCR Verification	Verify the positive clones via PCR	- Select colonies and perform colony PCR to check for phnE1/E2 genes	Positive PCR results confirm the presence of target genes
Day 10	Plasmid Extraction	Extract plasmids for sequencing	- Extract plasmids using a plasmid extraction kit - Measure DNA concentration	High concentration of plasmid DNA obtained
Day 11	Sequencing Verification	Confirm the correctness of the cloned genes	- Send the plasmid for sequencing - Analyze sequencing results	Sequencing confirms the correct gene sequence
Day 12	Strain Growth for Absorption Test	Prepare the engineered strain for absorption testing	- Inoculate positive clones into fresh LB media and grow overnight	Active strain ready for absorption test
Day 13	Prepare Media	Prepare LB media containing glyphosate	- Prepare LB media containing 80 mg/L glyphosate and 50 µg/mL ampicillin	Media is ready for upcoming experiments
Day 14	Initial Glyphosate Absorption Test	Verify the engineered strain’s glyphosate absorption capability	- Inoculate engineered strain into glyphosate media, take samples after 3 hours - Prepare samples for ELISA	Samples are ready for ELISA testing
Day 15	ELISA Testing	Measure the glyphosate concentration	- Use glyphosate ELISA kit to measure absorption levels	Absorption data obtained from ELISA

Day	Task	Objective	Steps	Expected Results
Day 16	Glyphosate Absorption Time-Course Test	Measure the time dynamics of glyphosate absorption	- Take hourly samples, centrifuge - Measure glyphosate concentration using ELISA	Data for the absorption time course
Day 17	Data Analysis and Optimization	Analyze time-course results and optimize conditions	- Analyze the absorption curve to identify the best absorption time - Optimize conditions such as shaking speed and temperature	Optimal experimental conditions determined
Day 18	Repeat Absorption Experiment	Confirm absorption efficiency under optimized conditions	- Repeat glyphosate absorption experiment under optimal conditions	Reliable and repeatable data obtained
Day 19	Construct phnE1/E2-phnJ Strain	Add the phnJ gene to the engineered strain	- Clone and transform the phnJ gene into the existing strain	New phnE1/E2-phnJ strain constructed
Day 20	Test Glyphosate Degradation Ability	Measure degradation efficiency of the phnE1/E2-phnJ strain	- Perform glyphosate degradation experiment - Measure degradation via ELISA	Glyphosate degradation efficiency data
Day 21	Construct phnO Strain	Clone and transform the phnO gene into E. coli	- Clone and transform phnO into E. coli	phnO strain successfully constructed
Day 22	AMPA Degradation Test	Test the AMPA degradation ability of the phnO strain	- Prepare crude enzyme extract - Measure AMPA degradation activity	AMPA degradation data obtained
Day 23	Repeat AMPA Degradation Test	Ensure reproducibility of results	- Repeat the AMPA degradation experiment	Stable degradation data confirmed
Day 24	Enzyme Activity Test	Measure the kinetic parameters of phnO enzyme	- Perform enzyme kinetics experiment with different AMPA concentrations	Km and Vmax data obtained
Day 25	Data Analysis and Report Writing	Analyze all data and start writing the report	- Analyze data using GraphPad Prism - Begin writing the experimental results and conclusions	Data analyzed, and part of the report drafted

Day	Task	Objective	Steps	Expected Results
Day 26	Construct Cold-Inducible Reporter Strain	Build a cold-inducible fluorescent reporter strain	- Clone the cold-inducible PcspA promoter and transform into the vector	Cold-inducible reporter strain successfully constructed
Day 27	Cold-Inducible Reporter Test	Test reporter gene expression at different temperatures	- Grow at 16°C, 25°C, and 37°C - Measure fluorescence and OD600	Temperature-dependent fluorescence data
Day 28	Construct Cold-Inducible Suicide System	Build a cold-inducible suicide system with the mazF toxin gene	- Clone PcspA-mazF and transform into E. coli	Cold-inducible suicide system constructed
Day 29	Suicide System Test	Test the suicide effect at cold temperatures	- Grow strain at 16°C and measure OD600 changes	Confirmation of the suicide system’s functionality
Day 30	Final Report Writing and Presentation Preparation	Complete the experimental report and prepare for the presentation	- Compile all experimental data - Finalize the report and prepare the presentation materials	Final report and presentation materials completed