The Identification and Characterization of a Halophilic UvrD Homologue, and its Incorporation into Helicase Dependent Amplification

Student: Kevin Miller
Major: Biochemistry & Molecular Biology
Minor: Mathematics, Environmental Studies
Advisor: Dr. Ben Leslie

Kevin MillerThis presentation is only available to College of Wooster students, faculty, and staff.

Abstract

View on Microsoft Stream

Kevin will be online to field comments on May 8:
2-4pm EDT (PST 11am-1pm, Africa/Europe: evening)

16 thoughts on “The Identification and Characterization of a Halophilic UvrD Homologue, and its Incorporation into Helicase Dependent Amplification”

  1. Awesome work, Kevin! I am curious: why are segments with high GC content hard to amplify with HDA?

    1. Thanks Dr. Faust! To answer your question, firstly, GC base pairs have three hydrogen bonds compared to AT base pairs which have only two hydrogen bonds which increases the strength of pairing between GC bases. But, actually even more so GC base pairs can participate more favorably in what is known as base stacking which stabilizes the DNA duplex making it harder to unwind or separate by heat. In fact, even in traditional PCR for segments with high GC content an additional “GC buffer or enhancer” will be added that contains DMSO or other PCR additives to improve amplification.

      1. Thank you for the explanation. I wish you the best at OSU, whatever your future research may be!

  2. Great presentation! Does the absorbance in the activity assay level out over time because the ATP is depleted?

    (P.S. I can see some great ways to follow this reaction by HPLC and HPLC-MS.)

    Thanks for presenting. Good luck in graduate and school and beyond.

    1. Good question. Actually, the leveling off in the ATPase assay is most likely due to the depletion of the MESG compound since it was at a lower concentration than the ATP in the assay. MESG is the compound that is converted to the product which is being measured at 360 nm. I agree HPLC could give some additional interesting insight and thank you!

  3. Nice work, Kevin! This is a cool project.

    Where are the various bacteria you looked at native to? Is O. antarctica native to salt lakes in Antarctica?

    1. Great question! Yes, O. antarctica was originally found in a deep salt see of Antarctica and is also considered a thermophile (adapted to conditions of extreme temperature in this case cold). T. oleivorans was actually isolated originally from a sea in Milazzo, Italy and O. messinensis was also isolated from a salt harbor in Italy.

  4. Hi Kevin–

    So proud of you for doing a virtual presentation at IS Symposium. It seems like only yesterday that you were in my environmental geology course, but that was spring 2018. Now, it is spring 2020, and you are on the doorstep of graduation. Now, I have to admit that while we are both scientists, your IS has some vocabulary in it that really made me press the pause button. My brain had to think!! Good on you for your BCMB major; I hope your next steps after graduation are fulfilling.

    Again, great job finishing IS, doing this Symposium, and for finishing finals this week. -SJ

  5. Thanks for sharing your research, Kevin! Towards the beginning of your discussion of results, you mentioned some potential reasons why you did not see comparable results between the NaCl and KCl solutions. If you had more time to revisit those tests, do you except the solutions would behave similarly or would the identity of the salt (NaCl vs KCl) make a difference?

    1. Thanks Dr. Craig, I would not expect too much of a difference between NaCl and KCl as really the only difference is the presence of sodium versus potassium which I would hypothesize have similar cationic effects on the proteins solubility. However, given that NaCl is more prevalent in the environment of these halophiles it may be possible NaCl is more likely to improve solubility. I am not aware of the extent to which research has been done on the interactions of proteins with sodium versus potassium. Again I think during my research I simply did not lyse the KCl cultures well enough to gather good data with respect to that question.

  6. Kevin, this work is so cool! If you could go back and tell your August self (or Junior IS self) something, what do you wish you had known going into the project?

    1. I think I would just remind myself that research takes a lot of trials and will most certainly not work the first time, but to just be patient. Protein over-expression took a lot more optimization than I anticipated!

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