Notebook
Summary of work done from January 2024 through June 2024, with weekly summaries from June 2024 through September 2024.
Summary of work done from January 2024 through June 2024, with weekly summaries from June 2024 through September 2024.
Ray, Lucas, Jael, and Troy at bench in Thomson Lab
The Notebook Folder contains a pdf of the Experiment Group's Benchling notes, protocols, and pictures.
https://tools.igem.org/uploads/teams/5035/lab-notebook
https://static.igem.wiki/teams/5035/lab-notebook/experiment-group-notes-1.pdf
https://static.igem.wiki/teams/5035/lab-notebook/iea-protocols.pdf
Inluded are the following protocols used in the enzyme design, and the expression classifier projects:
PCR Method
Purification(SPRI) Method
PURE (TXTL) Method
TXTL Method
Electrophoresis Method
TXTL Purification Method
Mini-Protean TGX stain-free Precast Gel Method
TXTL Under Denaturation Method
TXTL + Cofactors Method
Cofactor Reconstitution (Nakamura) Method
PCR Optimization Method
KAPA HiFi PCR Method
RepliQa PCR Method
Phusion PCR Method
GxL PCR Method
E-Gel EX 4% Protocol
Bioanalysis Machine Protocol
For the first months, we spent a lot of time (about 6-10 hours per week) on computation, learning and trying various techniques, refiniing computing skills, adapting, creating, and modifying software to design enzymes. We also learned new wetlab skills and protocols. When the summer started we spend a lot of time (10-12 hours per day) on computation (to create novel enzymes) and working on the expression classifier, because we discovered that novel enzymes are very hard to express in any circumstances.
Troy and Lucas Computing
In July, we had acquired most of the skills we needed for our wetlab work, and spent most of the time in the lab, in addition to computation work (10-12 hours per day, 5 days per week). All the wetlab work is documented in the attached Lab Notebook, and briefly summarized below.
Ray and Jael at bench in Thomson Lab
Intro: The main focus of the wet lab was to bring the dry lab’s vision and progress to life.
Throughout the first two weeks of the experimentation, progress focused heavily on the preliminary expression of candidate enzymes. This included DNA amplification, purification, and cell-free mixture expression. Additionally, results were measured through gel electrophoresis and Qbit analysis.
The third and fourth weeks of experimentation focused on the improvement of said expression as the preliminary results were inadequate. The basis of these weeks was expressing T7RdhA to see what was needed for the proper expression of main candidate enzymes.
The fifth and sixth weeks of experimentation were arguably some of the most tedious and strenuous of the project process. In these weeks, long processes of PCR and purification were completed for new enzymes in addition to T7RdhA.
The longest stretch of repeated and complicated processes came from the seventh through eighth weeks. During this time, the processes for enzyme expression became streamlined and thorough enough for results to be effective and efficient. The completion of PCR optimization, cofactor reconstitution, and electrophoresis analysis proved to be effective methods of expression.