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Overview

In order to record the daily wetlab progress and analyze the experimental results timely, we log the experimental contents for every experiment, and organize them. The compiled notebook is as follows.

Week 4, November 2023

Initial Planning for 2024

  • Deliver presentation on team introduction
  • initiate student recruitment process.
Week 1, December 2023

Initial Planning for 2024

  • Conduct recruitment interviews.
  • Finalize the selection of new members to the team.
Week 4, January 2024

Induction and Training of New Members

  • Conduct basic wetlab experiment related to synthetic biology and biochemistry.
  • Provide training sessions for drylab and model group members.
Week 5, January 2024

Induction and Training of New Members

  • Deliver theoretical knowledge on wetlab practices.
  • Continue drylab and model training sessions.
Week 2, March 2024
  • Collect brainstorming ideas during winter vacation.
  • Introduce CRISPRi as a potential component of our project.
Months of April - May, 2024

Concept Development and keep Brainstorming

Week 1, June 2024

Project Finalization and Pathway Design

  • Discuss the feasibility of Programmable Logic Device (PLD) as a target of our project design.
  • discuss about logic gates and recombinases as potential components of the design.
  • Initiate preliminary gene circuit designing around PLD, integrating CRISPRi, logic gates and recombinases.
Week 2, June 2024

Project Finalization

  • Engage in more detailed study of parts involved in our project.
  • Develop a feasible protocol and schedule upcoming wet lab experiments.
Week 3, June 2024

Plasmid Design

  • Complete the early stage of plasmid designs.
Week 4, June 2024

Fragment Design and Constitution

  • Source necessary genes. Design primers for plasmid fragment and recombinase genes
  • Assemble DNA fragments through PCR
Week 1, July 2024

Plasmid Construction

  • Carry out homologous recombination to construct recombinase genes and plasmids.
  • Transform the constructed plasmids into E. coli Top10 competent cells for propagation.
Week 3, July 2024

Patch Development and Innovation

  • Identify a flaw in the current design.
  • Develop and integrate a patch to fix the mistake.
Week 4, July 2024

Plasmid Construction and Validation

  • Utilize overlap PCR techniques to construct plasmid fragments.
  • Verify PCR results via agarose gel electrophoresis.
  • Recover fragments of specific length through gel extraction.
Week 1, August 2024

Problem Identification and Sequencing

  • Analyze and reflect on issues encountered in our design.
  • Fix the mistake using PCR and homologous recombination.
  • Sequence the plasmids to prove the success of modification.
Week 2, August 2024

Finalizing Plasmid Construction and Extraction

  • Re-integrate missing sections with the plasmids.
  • Extract plasmids and store them for future constitution and transfers.
Week 3, August 2024

Continued Plasmid Construction and Validation

  • Proceed with combining the Input plasmid and Register plasmids.
  • Utilize agarose gel electrophoresis to corroborate the results.
Week 4, August 2024

Orthogonality Verification

  • Pair six recombinases and three recombination-GFP sites to confirm orthogonality.
  • Transfer combinations into Top10. Promote cell incubation and growth.
  • Verify the accuracy of the orthogonal genes and recombinases.
Week 1, September 2024

Induction and Fluorescence Testing

  • Add IPTG and DAPG to induce the expression of recombinases.
  • Conduct fluorescence testing and agarose gel electrophoresis to certify inversion and excision.
Week 2, September 2024

Continued Plasmid Construction, Extraction and Transformation

  • Modify various plasmids and reconstruct them.
  • Extract complete plasmids for further usage.
Week 3, September 2024

Screening and Fluorescence Testing

  • Transform and conduct monoclonal screening and extend incubation time for successfully transformed E.coli.
  • Execute colony PCR. Verify the length of plasmids using agarose gel electrophoresis.
  • Carry out fluorescence testing with a microplate reader. Confirm the orthogonality of recombinases.
Week 4, September 2024

Continued Fluorescence Testing and Data Analysis

  • Continue fluorescence testing to confirm the orthogonality of recombinases and the functionality of register plasmids.
  • Add inducers for recombinases. Plot fluorescence-time graphs to analyze the effect of recombination.
  • Apply qPCR to verify the inversion and excision of the recombinases.
  • Design directed evolution experiments and collect initial results.
Week 1, October 2024

Wiki Freeze and Presentation Video

Week 2, October 2024

qPCR and Directed Evolution

  • Continue using qPCR to verify the modification ability of the recombinases.
  • Continue to conduct directed evolution for the efficiency of recombinase A118.
Week 3, October 2024

Plasmid Sequencing

  • Sequence the plasmid for further confirmation of the accuracy and functionality.
Week 4, October 2024

Grand Jamboree

Full records

For more detailed notebook description, please download the PDF of our lab record on benchling.