Douglas Reinisch, Weeks 6-7
My time at Hahnemann Hospital at Drexel University just came
to an end this past Friday, but not without making some great memories in my
final two weeks working in the Bouchard Laboratory. Both inside the lab and out, I tried to make
the most out of my limited time left in Philadelphia this summer.
The view of Hahnemann Hospital at Drexel Medicine from 15th Street.
My sixth week started off working on the new mutant sample
(HBx 50-131) that we had begun work on the previous week. Kennadi and I performed a QiaPrep MiniPrep
Spin Kit on the bacteria cells with our mutant DNA. Unfortunately when we used the Nanodrop One
to determine our concentration and cleanliness of our plasmids we found that
something either went wrong with the transformation or the MiniPrep. Afterwards, we continued to track the growth
of our HepG2 cells on the collagen plates until it was time for the weekly lab
meeting. Ron gave the presentation at
the lab meeting about his recent findings in his study of HBx’s relationship to
cellular glucose uptake.
Tuesday and Wednesday this week were very short days as we
were delayed due to complications with the microscope that is shared among many
labs in the building. On Tuesday we
redid the ligation and transformation for HBx 50-131 in hopes of correcting our
mistakes from the prior day.
Additionally, Joe wanted us to view the HepG2 cells at the proper
confluency as he planned to transfect them that evening (which he was not able
to). On Wednesday we made three gels (1
failed) for Western Blot analysis, which we were not able to run since the
HepG2 cells hadn’t been viewed yet.
Therefore, Joe took advantage of our added free time to go into depth
about the theory behind Western Blot and how important it was for our project. The Western Blot, live cell imaging, and
sequences were all yielded the most important results to our research in my
opinion.
Thursday was the last day for me in the lab that week as I
had a family event to go to on Friday.
We viewed the transfected, but not yet stained HepG2 cells under a high
powered microscope, allowing us to see HBx, thanks to the green fluorescent protein
that it was fused to. However, without
being stained with MitoTracker Red, we could not see the mitochondria to determine
whether or not there was mitochondrial localization of the mutant HBx proteins. Joe helped Kennadi and I work on our
presentations for our joint lab meeting we were presenting at on the following
Monday. Joe then took care of the live
cell imaging and running the Western Blot on Friday by himself as Kennadi too
was out of the lab.
My final week was much different than all 6 of my previous
weeks in the lab. After analyzing the
Western Blot that Joe ran on Friday, he helped Kennadi and I make last minute
changes to our presentations before our lab meeting at 2:00 P.M. He also questioned us on the material in our
presentations to make us more comfortable with our talking points. After a very brief lunch I gave the first
presentation and afterwards received a lot of constructive criticism from Joe,
Dr. Bouchard, and Dr. Stills to help me make my poster that I will be making
for EXP better than my PowerPoint presentation was. Kennadi then followed me in a similar
fashion.
On Tuesday, Joe and I split some HepG2 cells in what was a
shortened morning session. The reason
for this was that Joe, Dr. Bouchard and I had to Uber over to University City
side of the Schuylkill River to see Kennadi give her final presentation in
front of the other members of her program at the University of
Pennsylvania. We also had the
opportunity to listen to a few other students discuss their summer in various
research labs before we had to Uber back to Drexel.
On Wednesday and Thursday, work piled up a bit for me and
Kennadi not in attendance made it that much more for me to deal with. I began work on Wednesday by picking colonies
that Joe had transfected the previous day for HBx 50-131 and growing them up in
LB broth. I then made a couple of
Western Blot gels that Joe and I would run HBx-GFP on after lunch. The last thing we did was clean and autoclave
several dishes that had already been cleaned and autoclaved once before to make
sure they were completely clean as we were preparing to make 1M stock buffer
solutions of many different solutions. On
Wednesday, Joe and I both performed our own QiaPrep MiniPrep Spin Kit and
tested it with the Nanodrop One for the HBx 50-131 colonies that I had grown up
the prior day. Joe then did a
restriction digest on the samples with the highest concentrations of DNA while
I prepared the DNA gel we would run the samples on. The goal of this step is to make sure that
our mutant HBx plasmids are roughly the correct nucleotide weight, before we
send them off for sequencing. After
confirming this, we prepared and sent 4 of our 6 samples of HBx 50-131 off for
sequencing in time that Joe would get the results back before the weekend.
On Thursday I had to throw away one of our transfected samples of HBx 54-154, due to this unknown growth.
My final day at the Bouchard Laboratory at Drexel University
for the summer of 2017 was a bitter-sweet one.
Although I was saddened to realize that this would be the last time
seeing the faces of many people, being a part of the Philadelphia commuters’
community, and conducting firsthand meaningful scientific research, I was happy
to realize that I would have the rest of August free to do with as I please. Joe and I, (Kennadi took the morning off) analyzed
the Western Blot that we ran on Wednesday, for HBx-GFP wild type. However, before we did this we had to attach
the secondary antibody to the protein, which will allow us to view the protein
under the Odyssey CLx machine. Joe and I
then met up with Kennadi and two of her friends from her STEMPrep program for
lunch at Sabrina’s Café up in North Philly.
After this goodbye meal, Joe and I went back to the lab to autoclave
some dirty dishes that have been accumulated over my 7 weeks at the lab. It is pretty ironic that the last task I did
with Joe was one of the very first he had taught me 7 weeks prior. After saying my goodbyes I parted the
Bouchard Laboratory for what will probably be the last time.
Joe and I analyzing the Western Blot results using the Odyssey CLx machine.
Now that the research part of my EXP summer is over, looking
back over my time I am very happy that my prior labs either did not accept me
or dropped me unknowingly, because I truly believe that the Bouchard Laboratory
was the proper fit for me. With that
said, I could not be prouder to announce that because of my research, we have
narrowed down the region of HBx localization to mitochondria to the amino acids
54-131 region. This means that we have
narrowed down the region from 154 amino acids, to just 78 amino acids. Joe is going to continue with this research
until he narrows down the region as much as possible until he finds the region
that causes HBx localization and determines the effect that it has on HBV
replication. I now look forward to my
final year at Peddie and presenting my summer research to the Peddie community
in the fall.
The final time I emerged from the Patco station at the northwest corner of 15th and Locust Streets
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