Crystals and Clones

           The beginning of this week was spent finishing up working with the TOPO clones from last week. We preformed electrophoresis to determine which reactions contained the correct inserts. Nine of the sixty reactions produced positive results on our gels, which sounds pretty bad but Christine counts it as success. I am learning that things do not work most of the time and a low success rate is not anything to be afraid of. I having been doing a lot of cloning and DNA extraction this week to help the other interns prepare for their research projects that will involve looking into the role of environmental stress response in manganese oxidation.

           Some of the cloning I have been working on this week is attempting to put a His tag on MnxG. We did not have any success with TOPO cloning which may be attributed to the large size of the MnxG plus His tag fragment. We are currently researching Gibson Assembly, a DNA assembly kit that allows you to connect multiple overlapping DNA fragments to one strand (or into a vector in our case). My project will be working to make the long and arduous protein purification process more efficient by trying out various types of medium and tweaking the dialysis buffers to optimize protein expression. Currently we are culturing the cells in LB but I may experiment with SOC or other media. We got exciting news from the crystallographer that he was able to grow three protein crystals from the MnxEF sample we gave him several weeks ago. He sent us a picture and you can see the little hexameric crystals growing on a tiny drop of our purified solution. He will harvest these crystals (by hand using a tiny loop) and freeze them in liquid nitrogen to send them down to the synchrotron at UC Berkley. If all goes well, we will be able to go watch the him remotely control the synchrotron and collect the X-ray diffraction patterns that will be compiled computationally into a molecular structure of MnxEF.