In the Chilkoti lab, we work to create different proteins that could potentially improve drug delivery. To do this, bacteria must be constantly transformed, grown, and lysed. Each step takes hours, and depends on whether or not the previous step was successful. Because of this unpredictability, workdays can only be planned one or two days before. While each day may seem similar, every step is important for making a functional protein we can store and use for future purposes.
Some days we start at the beginning, with different pieces of plasmids we have to recombine. After cutting the plasmids using certain types of enzymes, we have a new piece of DNA that can be transformed into bacteria using a heat shock. These bacteria now have new plasmids that contain our desired protein sequence of ELP, and we allow them to create the protein as usual overnight or after a day. This step can take many hours, but necessary to allow for a greater yield of protein.
After a large amount of bacteria have grown, the protein must be extracted by lysing the cells. This erupts the membrane and allows the protein to be released into the surrounding solution. Afterwards, the desired protein must be separated and purified from other contaminants, which can be done by utilizing the ELP’s change in solubility under different temperatures. By switching between hot and cold temperatures and centrifuging the solution, the ELP can be isolated from other proteins that are always either soluble or insoluble. Through these cycles, the ELP becomes purified and ready to use for further testing.
It is important to note that these steps can be unsuccessful, and so a large amount of time is also devoted to checking our work and making sure we can continue with the purification. Without running gels to check DNA or protein size, we can’t be sure if the results we find are accurate. Although frustrating at times, the payoff of finding our sample to be purely our desired protein can make the days-long process worth it.
