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I am NOT a Bull in a China Shop!

This week, Jenn tells us about her pet viruses, dissects a publication, tells us what not to do with SDS-PAGE gels, and shares a bit of job-hunting advice.  Enjoy!

I’m beginning to wonder if I have some scar tissue on the part of my brain that helps me know my own strength. I may or may not have broken three (OMG 3) pieces of short glass while attempting to run SDS-PAGE protein gels (ahem) in the last week. I swear I’m not a brute! I never broke any short glass at PCC. However, I can definitely tell you three ways NOT to touch short glass because there is a very good chance it will end up broken.

Here’s a quick reference for you:
  1. Don’t try to push the two pieces of glass down into the holding tray with your thumbs on the short glass and your index fingers in the back on the longer glass. Trust me, it’s a bad idea.
  2. Pour resolving gel immediately. I’m talking hot potato immediately. When adding the comb NEVER exert pressure. Ease it in, GENTLY, and preferably at an angle to reduce the chance of bubbles in your wells. When doing this however, make sure you lined up the comb correctly in the first place because if you didn’t you may be tempted to exert pressure to get it in all the way. And what do we know about exerting pressure on the comb? We NEVER do it!
  3. When you are attempting to liberate the gel that was run, if you use a spatula, do NOT twist it like you would to break open a pre made commercially available plastic encased gel. That would be crazy! Instead, it’s a good idea to take the gel and let it sit in some running buffer or diH2O for about five minutes, then use the green separating tool or your hands to carefully, very gently, separate the two pieces of glass.
That last suggestion for how to separate the glass was carefully plead for on Facebook to the Bio-Link.org group page. STUDENTS: Join the group and pose your questions to the group! The combined knowledge of the people in this group is an amazing resource. Take advantage of their willingness to help you! (Finally, thank you very much to the person who made the suggestion. Sincerely, Eric Iverson) Just kidding. Sincerely ME, but probably Eric and Dr. Stedman too.
 

Since I’m on the topic of “Good to Know Info,” I would like to present a challenge that another undergrad student in the lab dealt with because I think it’s a great learning opportunity. (And I’m just really glad I didn’t do this first.)

As we search for internship opportunities and employment or volunteering opportunities we may get hyper motivated and find ourselves applying for well, everything. While this is a good idea, because (let’s face it) it is a bit of a numbers game, you have to find a way to keep track of your applications. Especially since in this field we have to tailor our resume for each position focusing on the skills we have acquired that specifically pertain to the opening and the employer’s needs. So, a few months ago, this undergrad applied for pretty much anything she was remotely qualified for at OHSU. OHSU is notorious for not even looking at applications unless you have a BS or BA. As such, she didn’t really expect to hear back, but thought of it as good practice and no harm done. To her surprise, she recently received an email inviting her to interview for one of these positions. The only problem was she didn’t know which position it was for or in which lab. She was able to do some detective work using the name of the individual who emailed her to backtrack and find out which lab had the position open luckily. Another thing she could have done, which I didn’t think of until the other day, was log into the OHSU system and see which of her application statuses had been changed to “Interviewing.” If that’s not an option though, and you get an email from someone in HR, I suppose it wouldn’t be too harmful to verify with the HR associate which position it was regarding. You would certainly not want to send that question back to the person in the lab who was inviting you to interview though. Moral of the story: Figure out a way to keep track of your applications! One way I’ve mentioned before is to use Huntsy. It is a little high maintenance though, so developing your own system would be a good alternative.

Now, what am I doing in the Stedman lab besides breaking short glass and ordering more online whilst doing the dance of my people to ward off the evil spirits of breaking short glass? Oh, you would ask! 

Well to answer that question, first we must talk about A genetic study of SSV1, the prototypical fusellovirus by Eric Iverson and Kenneth Stedman, published 05 June 2012, Frontiers in Microbiology. For your reference, a copy was attached to my previous blog entry or you can just click on the title above and it will take you to a page where you can get a PDF of the paper. Please DO reference it because I could be completely WRONG here. Grab some popcorn and get comfortable, but please don't start choking if you have to laugh at my interpretations of the information provided in plain English in the paper referenced above. 

Introduction:

In this research, three open reading frames (ORFs) were deleted to determine if the genes were necessary to the viruses viability for infection to the host, Sulfulobus Solfataricus, with the intention of providing more illumination on the functions of these ORFs (VP2, d244 and b129)

VP2 is the protein which represents the first ORF selected. This ORF is poorly conserved, meaning that it isn’t a gene which is always correctly carried into the next generation of replicated virus (at least I think that’s a good way to explain it).  It is suspected to be a DNA-binding protein although this is not yet confirmed. When this ORF is removed, the resulting virus is viable for infectivity and indistinguishable from the wildtype virus.  What my understanding about bacteria and viruses from the Microbiology class I took was that these guys very rarely carry unnecessary genetic material. So I have to wonder, why is it still showing up? If it’s poorly conserved, does that mean it’s probably on its way out of the virus’ genome? (These are student questions. Anyone feel free to chime in here to straighten me out.)

Next d244 which is a well-conserved ORF (that is believed to encode a nuclease) also produces a viable virus in the mutant resulting from this ORF being removed.

The last ORF studied in this paper is b129 which is universally conserved and predicted to be a transcriptional regulator results in a loss of infectivity when the ORF is removed and the resulting b129-less mutant is tested for viability.

The paper then goes on to give some additional information about the genome. It encodes a known thirty-four open reading frames (ORFs; Palm et al., 1991). Most of these ORFs have recognizable homologs within the family Fuselloviridae (Oh, I hope that’s a family. I think it’s Dear King Philip Came Over For Grape Soda – Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species.Yeah, so that's family then. The mneumonic device is priceless right? Thank you Dr. Dubbs! Back to what I was saying though…). However, they do not have any known recognizable homologs outside of Fuselloviridae, with the exception of one. The viral integrase encoded by ORF d355.

In this study there were three complementary approaches used to try to help determine the functions of these ORFs. These were structural genomics, comparative genomics and genetics.
 

Structural Genomics:

Using atomic resolution structures from C. Martin Lawrence, Iverson and Stedman were able to determine that among the three ORFs studied in this paper, b129 and d244 had homologs from other fuselloviruses. ORF b129 resembled transcriptional regulators (proteins that turn transcription, or genetic manufacturing from the blueprints to the protein, off or on).  ORF d244 resembled a homolog determined to be a new kind of nuclease.

Comparative Genomics:

Looking at the genome structure of SSV1, the most universally conserved genes are nearby each other in half of the genome, except the VP2 gene. Logic suggests that the highly conserved ORFs are needed for virus viability and function, but that has not been scientifically confirmed.  In my microbiology class we learned that these little creatures, like bacteria and viruses are pretty much all business, masters of efficiency. They don’t tend to carry a lot of extra genes around if they don’t serve a life sustaining or evolutionarily necessary purpose. Unlike humans who have all kinds of extra genetic material we haven’t gotten rid of yet. Then again, I could be making that up. No, I’m pretty sure that was discussed in Microbiology. Fascinating creatures!

Genetics:

Here is where the ORFs were individually cut from the plasmid, creating three different mutant versions of the virus. (I think.)

 
Materials and Methods:

Directly from the paper since it’s pretty clear on its own:
 

Sulfulobus solfataricus strains were grown on plates and in liquid media (YS). E. Coli was grown in LB at 37°C. 
(If you need me to explain this, you are most likely my Mom. We talked about Google already Mom.)
 

Plasmid DNA was purified from the E.Coli using alkaline lysis. The PCC BIT Program students did this a lot in DNA and Advanced DNA at PCC using the Qiagen kits. Iverson and Stedman used the GeneJet Plasmid purification Kit from Fermentas. After the DNA was transformed with SSV-(changed)d244 using the GeneJet plasmid purification, the DNA was retransformed (inserted into vector) E. Coli. Afterwards it was purified and analyzed by restriction endonuclease digestion with EcoRI (from Fermentas).

Here come the mutants! OK. A mutant of the virus is the result of the virus after the ORF is deleted using restriction enzymes and LIPCR. It's still the virus, but it has been changed. 

Directly from the paper again:
 

Deletion mutants were constructed from the pAJC97 shuttle vector using LIPCR (Clore and Stedman, 2006). Purified plasmid DNA was electroportated into Sulfolobus strain G theta as in Schleperetal.(1992)
 

The first strain of virus I cultured was the pAJC97 G theta strain. We observed these using the PSU Microscopy Core’s Transmission Electron Microscope. (More on that next week though.)

This brings us to the Discussion and Results sections. (Remember: NO choking!)

It appears that b129 is essential for virus activity. After repeating the experiment transforming b129 nine times they were unable to produce an infectious virus. According to the paper Iverson and Stedman cannot absolutely determine the ORF b129 is essential to virus function without additional experiments. According to other research though, by inducing the T6 transcript containing ORF b129 after UV irradiation seems to point to the ORF as predicted, being a transcriptional regulator.
An alternative hypothesis is posed that the b129 protein activates transcription of virus structural genes encoded by “late” transcripts T7/8/9, T1 and T2.

With regard to the SSV-changed d244, a still infectious virus was produced. It even produced clearer halos in the zones of clearing than those halos produced by the wildtype or the SSV-changed VP2 viruses. Additionally, when Sulfulobus infected with this strain was monitored for growth rate the data indicated that the growth was significantly inhibited. Here’s a fun mystery: when the changed d244 viruses inside of the Sulfulobus were transferred into new growth media multiple times the growth rates recovered to those similar of the wildtype virus. The changed virus was still present in these plates so the virus is not lost and does not have its coding rearranged. Additional speculation about the retarded growth noted by Iverson and Stedman include the possibility of the ORFd244 could be involved in the specificity of SSV1 integration. They are investigating whether or not SSV-changed d244 integrates into multiple positions in the host genome. Iverson and Stedman also propose the possibility that a defect in the changed D244 replication or resolution of SSV replication intermediates which lead to a bunch of bad DNA (they word it much more eloquently though), causing slower host growth. I feel like a 2 year old. Why? Why? Why?

Are you still with me?

Great.

While Eric continues to try to figure out what these ORFs are doing in the virus, one of my projects is to culture a couple of different strains to find one that is relatively homogenous in structure. Yours truly will be training in using the TEM (Most excited voice: DUDE FOR REAL!!!!! Have I mentioned my love for microscopy? Ever since I saw my first rotifer it was on.) to observe our little virus gems then package them up and send them to a Texas lab for cryo imaging.

I found lent gifts on my desk last week right before Dr. Stedman left for a trip. See…

I’m so excited!

Sincerely,

Jenn “I’m so excited!” Newsted
 
p.s. Regarding the cover pic for this blog: Don't act surprised. You could have guessed I have a panda phone cover. :)

p.s.s. Please, please, please add any comments of additional information helping explain this paper. For instance, I don't explain Long Inverse PCR because I did not get it. Anywhere I misunderstand or misstate the paper, please, please, please correct me! I appreciate your willingness to help us all learn!

p.s.s.s. As always, you can visit my website: jennifernewsted.wix.com/biosciencetech

Bio-Link Program: 
Portland Community College

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