One SSB protein from Bacillus anthracis does not the two binding modes!!
Students tested whether their SSB proteins wrapped DNA and if they have the two DNA binding modes similar to E.coli SSB. Experiments were done using a stop flow instrument where 1 uM SSB was rapidly mixed with 20 nM DNA containing a Cy3-Cy5 FRET pair. Wrapping = high FRET, formation of the differential binding modes is determined if there is a loss in the FRET signal. One SSB protein from Bacillus anthracis does not the two binding modes!!
6 Comments
Madyson Riddell
11/17/2015 02:28:56 pm
B. anthracis does not copy the same binding mode that E.coli does! This is very cool. In my research there have been hypothesis about a monomeric SSB for B.A. instead of tetrameric like in E. coli. I think this is a very important difference and a potential target for antibiotics! Also is there any potential for the salt ions interfering with the fluorescent tags?
Reply
Dr. Antony
11/23/2015 05:37:02 am
Madyaon,
Reply
Phil McFarland
11/19/2015 04:53:34 pm
I enjoyed this experiment because it gave us the ability to illustrate the dynamic binding modes of SSB. Using FRET to measure intermolecular tag distance on ssDNA is such a sophisticatedly simple method of determining binding orientation within SSB.
Reply
Dr. Antony
11/23/2015 05:29:46 am
Phil,
Reply
Nicholas Callard
12/4/2015 07:09:39 pm
Even though I wasn't in lab the day this experiment was conducted I was still very intrigued by the results that my group observed from the experiment. Our high salt condition only produced moderate FRET. While this was probably due to experimental error (lagging strand) there’s a possibility that the difference in Yersinia pestis's C-terminal tails and loops could be the cause of this difference in binding from E. coli. All the differentiations in the genomic sequence from E.coli were within the C-terminal tails. Knowing this might be a little optimistic, it still would be interesting to research further.
Reply
Tai Lu
12/7/2015 09:29:36 pm
This lab was very interesting in my opinion because with my friend Mike (who also has Yersinia Pestis), when we did this the outcome was not expected because the high salt condition only produced moderate FRET like what Nicholas Callard said. I also thought that it may have been an error as well during the preparation of the samples. Mike and I decided to retry the samples and when we got the results, it was still the same thing. That meant that we knew that it was not an experimental error. Overall I really enjoyed doing this lab. It gave us the ability to show the binding modes of SSB.
Reply
Leave a Reply. |
AuthorDr. Edwin Antony and Dr. Sofia Origanti has put together this course. The material presented here are generated by the students of the BIOL 4102 class at Marquette University. (Fall 2015). Improvements can only be made if there is adequate feedback and we thank you in advance for your time. Archives
November 2016
Categories |