Walter Hardesty Blog 3

     Hello everyone! The summer really has gone by so fast. I am so astounded by the amount of work that I’ve gotten done so far. There’s simply no way I could’ve done this during the quarter.

     I’ve spent the last few weeks trying to figure out why my carbonic anhydrase knockdown cell line is not exhibiting social motility at 5% CO2 during the SoMo assay. For a while, we all thought it was my technique. But now, we all think there’s something wrong with the cell line. After I showed the data to my P.I., he believes that I have kept these parasites in culture for too long.  Apparently, when you passage parasites (or any other microbe for that matter), in suspension culture for too long, they can acquire mutations or alter their gene expression levels in a way that can disturb normal knockdown levels upon RNAi (RNA Interference) induction with tetracycline. Wow, that was definitely a mouthful even for me! I guess the take-away lesson is that you want always want to be working with fresh aliquots of these parasites!

     Check them out! Notice the turbidity of the media in the flask. That’s a dense culture of parasites. Looks like it’s time to split (dilute) them into a new flask

 

     And also quickly, I wanted to introduce my good friend, Hoangkim Nguyen. She’s one of the graduate students who recently published a paper on CMF22, an axonemal protein that is required for propulsive motility in T. brucei. Kim is great! She has been a positive influence over my work here in the Hill Lab this summer. I come to her for advice on my projects, career plans, and life in general!

 

 



Walter Hardesty Blog 2

     Hey guys! I hope everyone is having an exhilarating summer both inside and outside of the lab. I’ve been working hard in the Hill Lab specifically with this social motility assay we like to call “SoMo.” I briefly explained it in my last blog and even showed a picture, but basically it’s a behavioral assay that tests the capacity for a trypanosomal cell line to engage in social motility. Because this is a new technique employed in the lab, we’re still working on optimizing the protocol. I’ve been the only researcher in our lab using this technique extensively as a way to answer my research question. For this reason, the graduate students, post-docs, and even my P.I. have oftentimes asked me for my input in terms of what I think works best. This is a great feeling because it underscores the importance of my contribution to the lab; it also reminds me that I am at the front lines of cutting-edge research. Other labs across the world including Switzerland are using this novel technique, so I assume it’s my responsibility to obtain robust and reproducible results!

     Anyway, enough of that. I’ve also been cloning in the carbonic anhydrase gene into a construct which contains an HA epitope. After my sequencing results improve (argh), I will soon be transfecting the parasites with the epitope-tagged construct and performing immunofluorescence to determine the localization of carbonic anhydrase.

Here’s me cloning.

 

So what you’re seeing here are a bunch of flasks containing different parasitic cell lines.

“IS THAT BLOOD?” one may ask

………

The answer is…….no! I wish, how cool would that be?

Actually, it’s just a pH indicator that turns red based on the pH range within the media inside.

 

 

 

Alyson Ramirez Blog 2

Hey guys!

So it’s been a while since I last checked in (sorry, I know you’re simply dying to hear about my latest experiences) and so many fun things have gone on in the past few weeks that it’ll be hard to keep things brief. I’ll do my best!

4^th of July: simply amazing. Photos below! We watched the best fireworks show I’ve ever seen in my life (timed perfectly to music) from behind MIT; the police presence was massive due to events earlier this year, and the show got pretty emotional. Boston Strong.

Research: things are going great! I’ve finished my first experiment, which was… completely inconclusive. But at least I know how to do in situs now, and I’ve been pretty self-directed the past two weeks while my mentor is in Spain- I know, I’m jealous of her too. I’m hoping to get more results from a second experiment next week, the fish have been reluctant to give me embryos (how selfish of them, right?) but I finally got things started this week. And I have to have a poster done in another two weeks. Where has this summer gone??

NYC: I travelled to the concrete jungle where dreams are made of last weekend. Wow. It was really hot and quite a change from the quiet, tree-lined streets of Cambridge. I ate some of the best food I’ve had all summer and ran around to all the places tourists flock to: Wall Street, the new World Trade Center, Staten Island, the Statue of Liberty, Central Park, Times Square, the Highline. And we did everything in one day! Needless to say my friend I was staying with was not amused with me dragging him around the city all day, but a few scoops of gelato made up for things. I also got to peek at a few of the schools I will be applying to in the fall for PhD programs, which has just made me more excited to continue my education.

Flour: I discovered this bakery down the street from where I’m living and any ideas of getting in shape/eating healthy this summer have officially been demolished. Just look at the picture of the sticky bun below and you’ll understand. I also woke up an hour early the other day to make sure I could get there for breakfast and still get to lab on time. Dedication.

Research seminars: Woah, people who work in this area are phenomenal scientists. We have weekly seminars where we get to meet the PI’s that everyone in my program is working with, and talk a little about their background. I’m completely awed by how everyone we have spoken with so far is so humble about their experiences, even though many of them have founded the institutions that they work in. Their dedication to translational research and finding solutions to the diseases that are so devastating to humans is really inspirational. It’s really made me consider why being involved in scientific research is important to me, and has made me more focused on my goals in the future.

There’s probably so much more that I could tell you about, but I won’t drag you through an unnecessarily long blog post. Until next time!

Adrian Hernandez Blog 2

Hi again fellow MARC trainees!

     I can’t believe the fifth week of the MARC-SPUR summer program is almost over! Currently busy studying for the GRE and taking the Princeton course offered by the SPUR program which has been a huge help. The latest workshops on how to apply for graduate level funding have been very insightful in showing other potential funding sources besides graduate level institutions. I have also started filling out secondary applications to several M.D.-Ph.D. programs and hope to send them out very soon.

     In terms of research, I have been busy studying the Rbfox proteins which are known tissue-specific splicing regulatory proteins. So far I have purified several Rbfox protein constructs which have deletions in the N- and C-termini to see if these mutations may affect RNA binding. I have also been trying to purify other proteins such as heterogeneous ribonucleoprotein F (hnRNP F) which is known to regulate splicing and mRNA stability and the KH type splicing regulatory protein (KSRP). I hope to simultaneously assay these proteins with an RNA substrate to see if there are possible protein-protein interactions that may affect protein-RNA binding.

Performing in vitro transcription to synthesize the radio-labeled RNA substrate.



Bacterial culture used for hnRNP F protein expression.

Adrian Hernandez Blog 1

Hello fellow MARC trainees!

      My name is Adrian Hernandez and I recently joined the MARC program this past spring quarter. That being said, the SPUR-MARC summer program highlights the beginning of my role as a MARC scholar. For the past two weeks I have attended and learned so much from the numerous workshops relating to research and graduate level studies. I especially found the workshop on personal statements to be insightful as I am in the middle of applying to several MD-PhD programs across the nation. I have also had the opportunity to meet other MARC trainees, as well as members of other summer programs who come from other states, like Tennessee.

     As part of my summer program I am continuing my research in alternative gene splicing in the lab of Dr. Douglas Black. Currently I am working on a side project dealing with the polypyrimidine tract binding protein (PTB) which is linked to the formation of the spliceosome complex for gene splicing. I am co-transfecting certain PTB constructs with reporter genes into N2A neuroblastoma cells to see how deletions in a specific linker region within PTB leads to altered splicing of the reporter gene during gene expression.

     I have been in Dr. Black’s lab for almost two years now and recently there have been some “changes” occurring as older post-docs pack up and head out to other colleges and universities to become principle investigators and run their own labs. Most notably I have been “asked” to move from my beloved bench and desk to foreign and not-to-distant lands.

On the left you will find my old, cleaned out desk. On the right is my new, cluttered and cramped space.

 

Here is a picture of my new lab bench. As you can see I have a lot more cleaning up to do.  

Taylor Brown Blog 4

Hello Everyone!

Sorry I’m late on my update haha; my labwork picks up and slows down, and now it’s slowed down, so I can write! Anyway, we got the sequencing results back! And I realized you may not know what colony PCR or a “miniprep” is…colony PCR is basically picking bacterial colonies and putting them, along with other reagents, into a machine that amplifies your DNA construct so that you can make sure it’s present by “running it on a gel” (gel electrophoresis). If you see bright bands around the size that your DNA should be, your DNA is there! A miniprep is basically a procedure you do after you have confirmed your DNA construct is present; you basically isolate the bacterial DNA from your colonies and purify it so that it can be sequenced. “Sequencing” is when you send your DNA off to a company so that they can provide you with the sequence of nucleotides (a long list of A,T,C, and G’s that code for your gene), and you compare your DNA results to the correct sequence that you were aiming for. Basically, the comparison looks like this:

 

except it’s much longer. The “subject” lines are part of the sequence for the gene that I want to study. The “query” lines are the sequencing results. You want a high Identity percentage, a low gaps percentage, and when you look at the long sequence you want to see lots of the vertical lines matching the letters in the subject and query lines (and no blanks where the vertical lines should be). So, my miniprep sequencing went well, and then it was on to a maxi prep! A “maxi prep” is basically like a miniprep, but growing up a LOT more bacteria. You start by taking a little bit of the bacteria that you used to do the colony PCR and the miniprep sequencing, placing it into LB broth (basically a liquid form of all of the nutrients the bacteria need to grow that looks like this:

(Photo credit to gbiosciences.com) and growing up a LOT of bacteria so that you can get a lot of DNA to work with (because as you know, bacteria replicate quickly). You know when you have enough a lot of bacteria because the flask gets really cloudy and smeeellllyyyyy! Haha I can’t describe the smell to you, really, but maxipreps aren’t my favorite thing to do; but they must be done! Anyway, then I purify the DNA again (with a procedure that I won’t go into detail about) and send it to sequencing one more time to confirm that I have the right DNA, and then comes the transfection! “Transfection” is when you take your DNA and viral components (the latter found in a liquid form in small tubes) and pipet them onto packaging cells in a dish. These cells then make the viruses for you with your DNA of interest, and then you put the virus onto the cells you really wish to study (called viral transduction), so that the virus can insert your DNA into your target cells. (My target cells are human breast epithelial cells called MCF 10As). But the whole process doesn’t just happen in a day, and it fails sometimes, so that’s what I’m working on now.

When you do virus work, you have to put on a space suit! Just kidding…but you have to put on a bunch of extra PPE (personal protective equipment) that includes: two pairs of gloves, a plastic gown with holes for your thumbs, and a face mask in addition to your labcoat (and long pants, socks, and close toed shoes). I modeled the process a bit for you below :) haha



And here's a picture of me in action :)



But anyway, looks like my cloning was a success guys! My mentor has another cloning project for me so that I can practice it some more, but looks like my first round of cloning went well! And I’m hoping my viral transfection and transduction worked so I can move on to my experiments! Thanks for reading; more info to come later!

Tay<3 br="" style="mso-special-character: line-break;"> p.s. …it’s my old man’s birthday today. Happy Birthday Papa! I love you(: