Madeline Dinsdale's blog

          In the lab this week I prepared more competent cells and cultured some more Mnx overexpressing cultures. I also did one last check on the Gibson assembly plasmids containing the Mnx full complex with a His-tag using an enzyme digest. The results looked promising and suggested that the assembly was successful! We will know for sure next week when the sequencing results come back.

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.

This week I worked on preparing plasmids containing MnxG and SOD(2) for sequencing from the TOPO clones we prepared last week. The sequencing data will be analyzed and used to confirm the presence of these genes so that the other interns can use those plasmids to investigate the effects of these proteins and others such as catalase and Oxy on thermal and oxidative stress response.

           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 have had a wonderful first week as a CMOP intern. I am working in Dr. Tebo’s lab, and my frontline mentor in Christine Romano, a post-doctoral researcher studying the biochemical mechanism of manganese oxidation in bacteria. This summer my project will entail working with her to purify and characterize the Mnx protein complex, a copper containing enzyme that is crucial for microbial manganese cycling.