All good journeys come full circle. After a week of preparation and cell harvesting, Dr. Masoudi and I moved on to setting up column chromatography with nickel resin – the first task we undertook together when I came into the lab last Tuesday. We made some slight modifications to the overall procedure, but it began just as it had before when we transferred incubated flasks with E. Coli cells to smaller centrifuge containers. After we spun our 12 liters of cell/media mix, Dr. Masoudi and I poured out the unnecessary liquid remains, combined the leftover cells, and added in a buffer solution to both maintain pH and limit biological activity. What’s interesting about E. Coli cells, and something I had not registered when we first did this two weeks ago, is that these “gram negative” bacterial cells have two layers of membrane. The first, outermost layer contains a periplasmic interior that carries our precious protein, Nb6B9, in addition to thousands of minuscule nutrients. The second, interior layer more closely mirrors the cell membranes of eukaryotes, outlining a cytoplasm which has a DNA-filled nucleus. In order to break the periplasmic layer (and not disrupt the cytoplasmic layer), we initiated an “osmotic shock” that fills the periplasm of each cell with enough water to burst the outermost membrane, expose the protein while keeping the cytoplasmic layer intact (… theoretically). The violent rupturing of the cells was followed by another centrifuge session, where bottles of the broken cells were spun at 14,000 rpm for about twenty minutes. The intense pressure delivered by the high speeds was able to segregate the intact cytoplasmic layers from the disrupted periplasmic contents, meaning we had to take the soluble fraction (the liquid fraction) of the bottles to properly retrieve Nb6B9. Unfortunately, our soluble fractions were viscous. The only macro-molecule capable of making the solution viscous, according to Dr. Masoudi, is DNA, indicating that we had ruptured the cytoplasmic layers of some of the cells and made the extraction process a whole lot harder.
The list of things I absolutely despise is quite short: spiders, Sauron, Dreamworks’ Minions. But the one thing that always tops the list, the one thing that punctures my soul and leaves me dangling on a mountain of turmoil, the thing that casts an infinite shadow over everything that’s good and innocent in this world, is bleach. The lab had run out of clean spin-bottles for the centrifuge, and Dr. Masoudi assigned me the task of washing out some used bottles under a specific protocol. As you may imagine, the risk of bio-hazards in the lab is really high, and therefore the risk of contamination between experiments is also pretty high. Cleaning ordinary, 250mL spin bottles is no “scrub-a-dub-dub” and your done. It requires determination, perseverance, and grit. You really have to grab the bottle by its sides and scrub it with the force of a thousand pounds of pressure. In other words, I rinsed the bottle three times, soaked the bottles and their caps in a bleach bath, and used deionized water to eliminate any chance of cross-contamination. However, the bleach’s horrible stench made my five-minute job seem like an infinitely long ordeal, but in hindsight, it wasn’t all that bad.
Just like yesterday, I helped Dr. Li Yin out by washing some of her flasks and pipetting exact amounts of protein into a gel electrophoresis machine – the plastic contraption that divides proteins based on their molecular mass. It’s been a pleasure working with Dr. Li Yin so far, and I’m honored to have witnessed the phenomenal research that she and Dr. Masoudi are conducing to obtain the crystal structure of the beta-2 adrenergic receptor bound to our infamous protein, Nb6B9. I am looking forward to what the rest of summer has to offer!
The lab is a peculiar place. There are professors, doctors, graduate students, undergrads, and high schoolers that speed through the halls, joke around, and take the time to get to know one another. It took me two weeks to realize that it wasn’t only the chemistry in our compounds that made the science interesting – it was the chemistry between people too. The lab is a living, breathing place that depends on scientists and interns who are willing to make sacrifices for each other, who are willing to go extra lengths to not only be good researchers, but good humans as well. These past two weeks have been a formative experience for me, and although the research is overwhelming and engaging, I can’t wait to spend more time with the people that make it all possible.
