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Allie Trafton
10/8/2015 05:25:36 pm
When I was first told that I was going to work with Bacillus anthracis (or anthrax), it kind of terrified me. But then I was told we were working with a protein not the bacteria as a whole. The first couple of experiments we have done, I have done before. This encompasses PCR, transformation of bacteria, and purification of DNA. This week is the week where everything is beginning to look a little foreign to me. But hopefully the new protocols will be explained well enough in lecture beforehand so that I won't be completely lost in lab. But with this week's binding experiment, is the ultimate reason why we didn't see two bands in the gels due to an error in calculated concentrations or in the amount of time we allowed the gels to run?
Dr. Antony
10/8/2015 08:09:34 pm
Allie,
Madyson Riddell
10/8/2015 09:18:10 pm
I have always thought that I hated lab because besides a freshman year Bio lab they have been Chemistry. Now I have to say while I still don't like chemistry labs, bio labs aren't bad! I especially like the continuity of the lab. We are working on a particular protein the entire semester instead of doing different experiments that don't relate to each other each week. I originally started work on Treponema pallidum, the bacteria that causes syphilis. I have little to no experience in a bio lab so pretty much everything has been a new experience for me. It's really interesting to see how these experiments are done and I like the logical continuation of things, like transforming a plasmid into a new bacteria and then checking to make sure the plasmid was actually transformed. Then once confirming the plasmid was transformed, checking to see that the plasmid contained the DNA segment of interest. It is so basic but not something I would have thought of.
Dr. Antony
10/9/2015 06:49:26 am
Madyson,
Netta
10/9/2015 12:53:50 am
I will be the first to admit Biology was never my strong point. When I began taking Biology labs last semester, I felt completely lost and inadequate in knowledge when compared to my peers. Chemistry is my life and mL, mols and liters are all I ever knew. When converting to Biology labs I had to get used to micropipettes and converting everything to microliters. I said all this to say, within the past four-five weeks, I've been the most comfortable within a Bioclass than I have within the past 3 years. This class helps me understand every procedure. Months ago no one could tell me that I'd be dealing with Meningitis SSB and figuring out its importance and relation to Ecoli SSB. The few things I love about this lab are the continuity. Each week, the experiments link to a higher level of understanding. In other words, every experiment builds on each other. I also enjoy the reading of research papers to incorporate into my lab reports. I never understood the purpose of certain lab protocols and the chemist in me never sought to figure it out either. However, these papers give me multiple interpretations of the SAME thing. Yes the information can be much at times, but it gives me multiple chances to understand why we are doing what we're doing. The functioning and purpose of certain gels over others. Like why we purified our proteins using SDS-PAGE over just pure agarose gel electrophoresis. The reason why we would see certain bands over others. So far Ryan & I have great success with our NM-SSB. We've been receiving FAT BANDS for our proteins and beautiful gels since day one. I hope that when we replace the Ecoli ssb with our NM-SSB there's an almost perfect complementarity. Also I'm excited to (hopefully) see the crystallized structure of our SSB. It's like watching a child grow into an adult (where they are free to be themselves, however you see all the work you put in). Too much? Lol I know! But I truly truly love the idea of working with one topic all semester. It helps me better understand structure and function of not only the organism I'm dealing with but also the differing methods I'm using as well.
Dr. Antony
10/9/2015 06:51:32 am
Netta,
Xinhang Yang
10/28/2015 08:27:44 pm
Our TP SSB protein only can be found on induced cell's band. If the problem is the protein degradation, I think it's better to purify TP SSB immediately just after cell lysis because TP SSB is not stable and easy to become fraction.
Dr. Antony
10/29/2015 06:37:40 am
For the TP protein, I'm afraid the problem is not protein stability. Its behavior during PEI precipitation is different than other SSB proteins. We should have seen the protein in the PEI pellet fraction, but it was mostly in the supernatant. Hence, we lost most of the protein in the wrong fraction. We could use other chromatography techniques such as affinity columns to purify TP SSB. At the time that we performed this experiment, it became readily apparent that the YP-SSB (Yersinia pestis) protein possessed a unique enough size, chemical behavior, and charge affinity which could be "exploited" for the sake of its separation from other biochemical materials. This experiment was striking because the gel illustrated each fractional step for the sake of comparison and qualitative analysis. Though SDS-PAGE is a novel experimental tool which can help users differentiate based on size, this experiment made it apparent that it is only useful if its user has a preliminary idea of what is to be expected based on the biochemistry. This is the novelty of the experiment in my mind, and this is what makes the YP-SSB protein purification so interesting. Without rehashing all of the details, the keys to this experiment in my mind involve our two additions once the YP-SSB was in (and out) of solution. Polyethyleneimine (PEI) is positively charged and forms complexes with dissolved YP-SSB protein which then precipitate out of solution. Since other negatively charged substances also precipitate, charge-based separation was followed by complex sized-based separation. This was achieved by centrifugation, leaving YP-SSB in solution, followed by the addition of solid (NH3)2SO4 to once again precipitate it back into a pellet which we ran on the gel. It is fascinating how this lab felt like an organic chem lab more than a biology lab, because the biochemistry was essential to our YP-SSB-specific purification. It was also exciting to discover the similarities our YP-SSB shared with E. Coli SSB's already published purification strategies.
Tai Lu
12/7/2015 09:49:30 pm
When I heard that I was going to be using the bacteria Yersinia Pestis, I didn't know what this type of bacteria causes until I did further research, learning that this bacteria causes the plague. From what I know about the plague, the Black Death was the last time I heard about the plague as it killed millions of people in Europe a long time ago. That really terrified me because it is dangerous in my opinion. 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
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