The final countdown…

I am sad to report that my ten-week fellowship is coming to an end. With the end of the summer fellowship comes an end to my blogging experience. My project will continue during the fall semester; however, I will be a little less flexible with my time to report on account of homework and the like.

As of late in lab, I can pretty safely say that one point mutation is not the cause of temperature sensitivity in therep gene in B. thailandensis. The best guess at this point is Ts can be attributed to a combination of mutations or quite possibly all five in the specific orientation are needed for the necessary tertiary structure of the Rep protein.

As far as the next step in my project: I have created and ordered new primers to change the fully mutated temperature sensitive plasmid back to the wild type rep sequence one point mutation at a time. This process is the reverse from the original method I employed. Stratagene’s QuikChange Multi Site-Directed Mutagenesis Kit will be used to convert one amino acid per PCR reaction from mutated to the wild type. (If you can recall: there are five different amino acid changes in the rep gene that were said to cause Ts in Burkholderia thailandensis)From this point on the entire process follows the same protocol as I have been doing the last ten weeks.

Once the PCR mutagenesis is complete, a digest and transformation into ultracompetent cells will take place. From here clones will be sequenced for verification of “mutation”. Plasmid DNA will be isolated and electroporated into B. thailandensis. Once colonies appear on selective media, clones will be patched for screening of temperature sensitivity.

If Ts is still seen in one or more of the clones different mutations will be changed back to try to further delineate the exact sequence necessary for Ts in B. thailandensis.

As well as taking this approach, I am also trying to create a library of combinations to burn the candle at both ends. Multiple plasmids containing different combinations of the five mutations were pooled and electroporated into B. thailandensis. Colonies will be patched and screened for Ts. If I am so fortunate to find clones that do in fact exhibit Ts, these clones will be sequenced to determine which combination of mutations leads to Ts in B. thailandensis.

Although I have not been successful in a “crossing the finish line” standpoint, I am hopeful that with time a more clearly defined sequence will be discovered. I know this is a tad cliché but, it’s like Aesop’s fables always told us, “slow and steady wins the race.”

With that I thank anyone who has been able to read and keep up with my posts and I wish you a happy end to summer and a hopeful end to the decade.

Go in peace,
Erin Breland

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