Week 1 Part 2: Hit the Ground Running

I started doing lab work on Wednesday. I am surprised by the amount of independent work I am doing already! My PI has the following policy: if there is a simple document explaining the step-by-step process, she will show me the document and I should do the work on my own. If the work is more complicated, she or her research assistant Wren will show me once and then watch me as I do it to make sure I’m alright. Afterwards, I am on my own. Thanks to this policy, I have done approximately 2/3 of the week’s lab work independently (with minimal to no supervision).

Before I explain what I did this week, I have to explain my project! Bacillus subtilis, under anaerobic conditions, can switch from aerobic metabolism to anaerobic nitrate metabolism. This process is regulated by the ResDE signal transduction pathway. Currently, we do not know the identity of the signal ligand that ResE senses, but Michiko suspects that it may be one of the terminal aerobic oxidases. So, I will be doing the work to find out if terminal aerobic oxidases are involved in ResDE signal transduction.

The first step is to quantify the effect of mutated (non-functional) terminal aerobic oxidases on ResDE signal transduction. I can monitor levels of ResDE signal transduction by measuring β-galactosidase activity in strains that carry the ctaA-lacZ expression reporter. The experimental method I am using is called the ONPG/β-galactosidase assay (shorthand: lac assay).

This week, I finished a whole set of lac assays. On Wednesday, I made a bunch of solutions for my personal use, made plates, and streaked them with my desired strains. On Thursday, I used the plates to inoculate liquid cultures. I put the culture flasks in aerobic conditions and took samples every hour to measure absorbance at 600 nm (a measure of cell density) and to freeze for later testing. On Friday, I took the saved samples and measured β-galactosidase activity. It was hard to do things on my own, and I made some mistakes. However, I have learned from my mistakes and I feel proud that I have learned my way around the lab so quickly.