Ending the semester on a high note!

Before winter break laziness (and hopefully some reading/writing energy) fully take hold, I wanted to report on a discovery from the end of the semester that will definitely be a focus of my project in the spring. The J75 cag negative strain of HP has never been the most cooperative. After going through several rounds of mutagenesis reactions and several primer sets, I finally altered the oipA locus to “phase on” and transformed this plasmid into HP. But when June and I started using these strains in adherence assays with AGS cells, the results were, frankly, boring. Unlike the other cag negative strain J68 where there were clear differences in AGS cell attachment between the cells with oipA phase on and off, there was no difference in J75 across several experiments. After confirming that each strain was indeed what it was supposed to be with sequencing, I was perplexed.

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Experimental Phase of “The Curious Case of OipA”

Hello and welcome to the wrap up of the Fall 2016 edition of “The Curious Case of OipA!” Since I was last on the SuRGe blog, I have completed phase 1 of my project, and have successfully created all of my mutant oipA strains of Helicobacter pylori. Now I am well into the experimental phase, and have some interesting preliminary results regarding the role of OipA in the pathogenicity of both cag pathogenicity island negative (less virulent) and positive (more virulent) strains of HP.

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Future Directions for “The Curious Case of OipA”

While we have made a lot of progress in the project this semester, there is still very much work to be done. Currently we have J75 and 26695 with BamHI sites digested as well as PMM674 (which has the CAT-rdxA cassette). Next step: run a gel with a small amount of digestion of J75 and 26695 just to make sure they cut correctly by comparing to the uncut plasmids with the BamHI site. Simultaneously, run gel on all of the PMM674 digestion to purify the CAT-rdxA cassette. Then ligase the two together, transform into E. coli, extract plasmid, and then confirm that the CAT-rdxA cassette is inserted correctly!

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Spring 2016 Progress

This was my first semester back in the lab since last spring- very exciting! Since I have been back from studying abroad, I have been starting work on what will be my honors project, “Turning on oipA in cagPAI negative strains of Helicobacter pylori,” or, “The Curious Case of oipA” as I like to refer to it.

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