One word: contamination. It’s like muttering the dark lord’s name. It’s like cursing in front of your mother. You say it and the whole world crumbles around you. The E. Coli flasks that Dr. Masoudi, Dr. Li Yin, and I had put together did not show growth over the weekend, meaning that some sort of alien substance had ousted our benign bacterium. Initially, Dr. Masoudi thought that the unwelcome stranger was Phage, a horribly persistent virus that would require a thorough bleaching of our workbench, but something had caught his eye before we went to bleach Dr. Li Yin’s station. At the bottom of our LB medium, a solution that we evenly poured in each of the E. Coli flasks, there grew an innocuous speck of fungi that competed for nutrients against our bacteria. It was this seemingly minor contaminating agent that forced us to pour 12 liters of prepared bacterial solution down the drain.
After washing our flasks and going over protocol again, we had to prepare new bacterial colonies that will house our precious Nb6B9 protein. We made two separate solutions: the first solution contains our LB medium and agar, a solidifying agent, while the second solution only has LB. The first solution was poured into about twenty petri dishes that serve as the houses for our bacterial colonies; we followed this by adding in the antibiotic kanamycin. Colonies will form overnight once we introduce E. Coli cells that have a particular plasmid, a set of DNA that is both resistant to our antibiotic and has the target gene for Nb6B9 production. We ensure that the plasmid enters the cell envelopes of the E. Coli with a half-hour cooling process followed by a prompt “heat-shock” that loosens up the cell membrane, increasing it’s permeability. Antibiotic exists in the petri dishes to weed out the bacteria that won’t produce Nb6B9 at our desired capacity, leaving the successful colonies that we will grow and eventually harvest. It’s a long process, but it’ll be rewarding once we extract our precious protein.
In the meantime, we checked up on the results from our Western. The membrane that has our protein imprint was taken to a special scanning device that emits different wavelengths of light to expose the presence of different bio-molecules. For example, DNA is detected using short-wave ultraviolet radiation. We had proteins though, and we forced the molecules to react with a substrate that leaves a bio-luminescent product. We turned the scanner from an ultraviolet machine to something like a photographer’s “dark room” so that we could see the faint bio-luminescence. What was found was a single strip of protein between 7 and 12 kDa that matches the identity of our Nb6B9 protein, indicating that Nb6B9 didn’t form any dimers (combinations of itself) and can be filtered out when using carboxypeptidase (the enzyme that we used to differentiate Nb6B9 from its constituent peptide chains).
