Week of July 17-23
On Monday morning, we began round two of the preservation protocol, as our negative controls (finally) remained negative! We transformed the DNA for the interlab study and TodX, BenzABCD, and the upper operon. Also in the wet lab, we ran the lower operon gibson assembly in a gel, prepared inoculation colonies for SDS-PAGE, miniprepped the lower operon, and began the next MIC50 assay iteration. In preparation for the lungs, we emailed potential contacts about gaining access to a vibratome or microtome and performed a dry run/control of the DNA extraction, PCR, and gel for the lung swabs. In the dry lab, we purchased a new website domain with Jimbdo and worked on the model. We implemented competition into the mathematical model and created separate growth functions for different types of model.
Tuesday we plated the first day of the preservation protocol, worked on transformations for the interlab study, prepared competent cells, resuspended the primers, miniprepped the upper operon twice and he lower operon and GFP, inoculated gBlock 4, and performed a transformation. We also got the lungs! In preparation, we cleaned out and set up the mammalian cell culture incubator, attended a vibratome training with the Bartlett lab as they sacrificed a rat for a brain study and sliced the brain with the vibratome, and made a solution of GFP-transformed E. coli and PBS buffer. We attended the euthanasia training lab and obtained rat lungs from the necropsy of the animal. We returned to Bindley to swab the lungs, lavage them with the E. coli solution, ventilated the lungs, and swabbed them again. We attempted to use the vibratome in different methods with the aid of the Bartlett lab to no avail. In the dry lab, we introduced competition into the model for the engineered bacteria and the native bacteria and tested the model’s ability to handle calculations for multiple days with the new methods.
On Wednesday, we continued the preservation protocol, and performed PCR on, ran a gel of, excised, and performed gel purification on gBlocks 1-3. We also digested GFP and the lower operon, transformed gBlock 4, and ran a gel on the Gibson assembly of gBlocks 4-6, TodX, BenzABCD, the lower operon, and GFP. We prepared for the MIC50 assay, the third iteration of which was started on Thursday. Finally, in the dry lab, we continued working with the model, implementing competition with benzene degradation affecting the growth rate of cells.
Thursday began with continuing the preservation protocol and performing a miniprep on GFP. We also performed DNA extraction and PCR and ran a gel on the pipette tips which had been used to swab the lungs, worked on the MIC50 assay, and ran a gel on gBlocks 1-3, excising the correct bands. In the dry lab, we added TodX to the mathematical model and performed a literature review on benzene uptake in the lungs and relative benzene degradation due to lung biome metabolism. At the end of the day, we had our weekly lab meeting with Dr. Solomon.
Friday started with continuing the preservation protocol and inoculating upper operon colonies. We minprepped GFP and prepared it for digestion. We loaded the digestion mixture and excised bands
and prepared for the interlab study work to be done on Monday. In the dry lab, we added competition, growth, benzene degradation, and enzyme kinetics to the mathematical model.