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They Grow Up So Fast

This week was spent growing up massive E. coli cultures for cell harvest, which will eventually be used for more Mnx protein purification. This process of making competent cells to transform with the pASK vector that contains either a full manganese oxidation complex (MnxDG) or a partial complex (MnxEF), then growing the cells up, harvesting them at the optimal time for protein expression, then purifying the protein is long and complex. You need to pay close attention at every stage of culture growth because man do these babies grow up fast. The doubling time of E. coli is every half hour, which means that culture density increases slowly at first then very rapidly. Before you know it they are ready for protein production and harvest, and if you don’t stay on top of it they’ll start running around with the wrong crowd, having unprotected conjugation and dividing like rabbits. I’m joking of course, but for real it is very easy to let the culture overgrow and miss the optimal culture density.

 I am finally getting the hang of the whole process, and this week I did it on my own with only the help of the high school intern. The cell harvest process is very technical and you have to be careful to time everything exactly. We start by inoculating 8 1L cultures with MnxDG and MnxEF and let them grow several hours until they reach the desired optical density. Then we remove the MnxDG cultures and put them on ice until the culture cools to 17C, the optimal temperature for the Mnx full complex to be induced. We add ATC (Anhydrous Tetracycline) to induce protein overexpression. Once the full complex cultures have been induced, they are placed back in the incubator overnight. The partial complex EF cultures are treated slightly differently. Once they reach the desired optical density, the ATC inducers is added while the cultures remain at 37C because the partial complex induces best at a warmer temperature. The Mnx complex contains copper, and we have to be careful to add sufficient copper at every step to keep the protein “fully loaded” so that it can successfully overexpress. When prepping the cultures for harvest we want to make sure they are fully loaded, so we add CuSO4, then let them sit overnight. The next day we harvest the cells by spinning them down in a centrifuge. The end product is about 15 mL of cell pellet, which is about the same color and consistency as peanut butter. This is flash frozen by submerging the tube containing the “cell-butter” in liquid nitrogen and then stored in the -20C freezer until we are ready for protein purification. My mentor, Christine, has done a ton of work on this protocol to determine the exact right conditions to optimize the process. It’s crazy how much cell mass you need to procure even just a tiny amount of protein.

            The bulk of my time in the lab is aimed toward further optimizing this protein purification process. One way that I have been working on this is to make a pTRC vector that contains a His tag and a full MnxDG complex insert. The His tag system is a way of molecularly tagging the Mnx complex with a polypeptide “tail” consisting of six histidine molecules. The current protocol uses a pASK vector with a Strep tag system, but the His tag has several advantages, though it is harder to assemble. The His tag is a mental chelator, meaning that it will bind free floating metals in solution. This may be a problem became out protein is an organometallic complex, but Christine thinks that it may also prevent some of the non specific copper binding we are seeing by removing some of the metals in solution. In order to make this MnxDG + His tag vector I used Gibson Assembly then transformed the assembled plasmid into competent cells and cultured them overnight. I preformed a PCR to confirm the presence of the MnxDG insert in the transformed bacteria. The PCR gel check was inconclusive, so I will do a restriction enzyme digest check next week. I really hope the restriction enzyme digest shows that the assembly was successful!