Image

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.

Week 4, November 2023

Preparation for 2024

  • Lecture on introducing our team and recruiting new students.
Week 1, December 2023

Preparation for 2024

  • Recruitment interview.
  • Determine new members of WetlabWetlab.
Week 4, January 2024

New members education

  • Basic wet-lab experiment about synthetic biology and biochemistry.
  • Drylab and model training.
  • Meet with PI at iBP.
Week 5, January 2024

New members education

  • Theoretical knowledge of wetlab.
  • Drylab and model training.
Week 2, March 2024
  • Collect and brainstorm ideas during winter vacation.
  • Introduce CRISPRi as a probable part.
April - May, 2024

Brainstorm

Week 1, June 2024

Project Determination and pathway design

  • Introduce the programmable logic device(PLD) as a probable design.
  • Introduce logic gates and recombinases as probable parts.
  • Design a preliminary gene circuit of PLD combining CRISPRi, logic gates, and recombinases.
Week 2, June 2024

Project Determination

  • Further investigation of parts in our project.
  • Find feasible protocols for wet-lab experiments.
  • Scheme the upcoming wet-lab experiments.
Week 3, June 2024

Plasmid design

  • Designed an early version of plasmids.
Week 4, June 2024

Fragment design and constitution

  • Acquire genes from various sources.
  • Design primers for plasmid fragments and recombinase genes and order primers from a reliable supplier.
  • Fragment constitution using overlap PCR.
Week 1, July 2024

Plasmid construction

  • Homologous recombination is used to build up recombinase genes and plasmid construction.
  • Transformation, colony PCR, and sequencing
Week 3, July 2024

Patch update and improvement

  • Discover a flaw in the current design
  • Build a patch to avoid the leak.
Week 4, July 2024

Plasmid construction and verification

  • Use overlap PCR to build up plasmid fragments.
  • Verify PCR results through agarose gel electrophoresis.
  • Regain specific length of fragments by gel extraction.
Week 1, August 2024

Problem-solving and sequencing

  • Reflect on the problems that emerged during the experiment processes.
  • Improve some fragments in the plasmid design.
  • Test the feasibility and send for sequencing.
Week 2, August 2024

Plasmid correction and extraction

  • Reunion missing sections with the plasmids.
  • Extract plasmids and temporarily store them for further constitution and transfer.
Week 3, August 2024

Plasmid construction and verification

  • Continue to combine the Input plasmid and Register plasmids
  • Agarose gel electrophoresis to confirm the result.
Week 4, August 2024

Orthogonality verification

  • Six recombinases and three recombination-GFP sites were paired to prove the orthogonality.
  • Transfer couples into Top10. Incubate and cultivate cells.
  • Verify the correctness of the orthogonal genes and recombinases.
Week 1, September 2024

Induction, fluorescence testing, and sequencing

  • Add IPTG and DAPG to induce the expression of recombinases
  • Conduct fluorescence test and agarose gel electrophoresis to prove the inversion and excision.
  • Examine the results. Send bacteria for sequencing.
Week 2, September 2024

Plasmid modification, extraction, and transformation

  • Modify several plasmids and reconstruct.
  • Extracted integral plasmids.
  • Couple recombinase with register plasmids and transform each couple into E.coli.
Week 3, September 2024

Screening, incubation, and fluorescence testing

  • Conduct monoclonal screening and extend incubation for successfully transformed E.coli.
  • Proceed with colony PCR. Verify the length of plasmids using agarose gel electrophoresis.
  • Test fluorescence with a microplate reader. Verify the orthogonality of recombinases.
Week 4, September 2024

Fluorescence testing and curving.

  • Continue fluorescence testing to confirm the orthogonality of recombinases and the availability of register plasmids.
  • Add inducers for recombinases. Draw fluorescence-time curves to examine the effect of recombining.

qPCR and directed evolution designing.

directed evolution of A118

Full records

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