Pacific Bacterial Genomics July 11th: A Trying Transformation

My day began, as it usually does, with breakfast at 8:40 am. I had my usual meal, minus the yogurt which, as I discovered, had apparently succumbed to the basketball camp participants probably long before I arrived. 

After breakfast, we headed to Strain Hall to begin our longest lab yet: natural transformation in V. natriegens. This experiment essentially tested whether our hypothesized antibiotic-resistance gene could be transferred to a wild-type strain of V. natriegens through the uptake and incorporation of foreign DNA into its genome. To confirm a successful transformation, we plated the transformants on antibiotic plates and compared them to control cells on non-antibiotic plates to calculate transformation efficiency (i.e., the percentage of cells that successfully acquired the antibiotic resistance marker). 

That all sounds dainty, except the problem is that the gene we PCRed didn’t mutate in any of our antibiotic-resistant strains. So going into the lab, we knew that in all likelihood, our natural transformation wouldn’t work. Unfortunately, we won’t know the outcome until tomorrow, so I apologize for the cliffhanger. 

Anyway, we proceeded to set up the transformation in the lab using the overnight culture (containing a mutated strain with a pMMB-tfoX plasmid) that our TAs kindly prepared the night before. This took about 30 minutes. Here are my dear labmates Aarjav and Violet pipetting our culture into the vials:

After finishing the experiment’s set-up around 9:30, we incubated the vials and proceeded back to the lecture hall to continue with data analysis, creating figures, and designing our poster. 

Soon enough, 12:00 rolled around, so we made our way to lunch. Luckily, we arrived at the cafeteria before the basketball campers arrived, so the line was mercifully short.

The conclusion of lunch reined us back into our natural transformation saga. We started in the lecture hall where we briefly discussed the history of horizontal gene transfer in bacteria to establish the context for our transformation experiment, then briefly returned to the lab to add LB3 broth to our vials and place them in a shaking incubator. 

This took approximately 5 minutes, so before we knew it, we were back in the lecture hall for more paper and poster work. 

Around 3:00 pm, we returned to the lab to complete the remainder of the experiment. It was my first time performing drip plating, so it was a bit difficult to get the tilting right to avoid cross-contamination at first. We also used a record number of microcentrifuge tubes so far, totaling in at 28 (we used 7 tubes in the serial dilution for each of three mutated strains of V. natriegens plus one strain of P. leiognathi). Tomorrow, we’ll check on the plates to see if our transformed bacteria (and our negative control too, I suppose, but hopefully not) grew.

Dinner ran as usual. Following a short break when I worked on the figures and this blog, my group and I convened for study hall. Now, we wait in bated anticipation for the results of our natural transformation lab. 

Thanks for reading!

Cyrus Ghane