Hustling to make it to the lab on time, I ran into Dr. Masoudi. We had a short discussion about our protocol today and we soon proceeded to get some E. Coli cells that were injected with a specific compound known as “IPTG.” This special protein turns on the promoter for a gene in E. Coli’s DNA that produces a nanobody called Nb6B9. Needless to say, I was utterly oblivious this morning. It took me a while to better understand what exactly was happening, but when I got a hang of things we started rolling. We began by vigorously shaking the bottles of E. Coli using some machines that bounce up and down upon contact with the bottle; my hand was numb after using this “vortex machine.” After the E. Coli had been disrupted enough and mixed with some buffer solution (basically a stabilizing solution), we placed the bottles in a freezer room to prevent any unwanted reactions. But Dr. Masoudi and I soon came across a pretty severe problem. The E. Coli bottles were filled with foam that made it nearly impossible for us to extract the cell solution. Usually, this wouldn’t be such a major issue, but the next step in our procedure was to use a gargantuan centrifuge (giant spinning device). As the bottles rotate at 14,000 revolutions per minute, the pressure against the sides of the bottles greatly increase and any space not occupied by dense solution (any of the area with foam) increase the risk of collapsing the bottle, ruining the centrifuge, and costing the lab tens of thousands of dollars. Did some famous person say something along the lines of “successful people take risks”? Because that’s exactly what we did. We balanced a couple half-filled bottles and popped them into the centrifuge, hoping to the gods of science that we wouldn’t hear an explosion. The centrifuge continued spinning for a good ten minutes without any problems, so we used the same procedure for the rest of the bottles. The well-mixed E. Coli solutions were poured altogether in the same flask, but another problem arose: the liquid was viscous. Because we needed to filter the E. Coli mixtures through a paper with fine, minuscule openings, a viscous liquid that binds together powerfully wouldn’t be able to meander through the holes. Only after adding a special enzyme, benzonase, did we have a watery liquid that we could us in our setup.
In the image above, the blue part of the column is nickel resin that binds to our oh-so precious protein, Nb6B9. The filtered liquid was dripping at a rate of about 2mL per minute, meaning the entire process took up to four hours.
The greatest lesson I’ve learned from my first two days in the Lefkowitz lab is that understanding is a requirement for appreciation. I thought I had a good handle on chemistry before I got into the lab, but I’ve learned that there is so much more to know, so much more to analyze, and so many more opportunities to take risks.