The Next Phase of My MK-STYX/Autophagy Summer Research

Hello Everyone!

After concluding some exciting work on MK-STYX and its impacts on autophagy organelles, I will now turn my attention to how our lab’s protein of primary interest, MK-STYX, may impact the production of the essential autophagy component LC3-II.  This protein is essential to the generation of the signature autophagy vesicle known as the autophagsome, so quantifying a change in its cytosolic expression after activating MK-STYX would strengthen our hypothesis that posits MK-STYX as a significant player in the autophagy pathway.  Additionally, I will be working closely with my inspirational autophagy project teammates Emily Pickering and Y-Nhui Bhui to characterize the nature and extent of MK-STYX’s activation of autophagy-promoting transcription factors.  We will analyze how the localization of these crucial polypeptides may shift using Cell Profiler computer algorithms manual scoring.  I will post an update in a few days about my progress with the aforementioned topics and my research plans for the rest of the summer.

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Troubleshooting is a pain, but a worthwhile one…

While running my 3rd and 4th trials for my experiment on TFEB localization in the presence of the pseudophosphatase MK-STYX, I have noticed that all my cells have died and that I will need to re-run my 3rd and 4th experiments in order to get data. The fact that my experiments have failed once again was disappointing and coming to terms with the fact that I will have to spend at least a couple more weeks before I can get my data been tough, but I have come to realize that the only way to get over this hill is to sit down and troubleshoot. I went back into my notebook and looked over every step that I performed and hypothesized what might have gone wrong during my execution of these steps. It turns out that I have forgotten a small but very important step: trypsinizing my cells. Without proper trypsinization, HeLa cells continue to adhere to the flask and are unable to be transported into a six-well plates during seeding. The cells that I have obtained from the flask without trypsinization were not live cells to begin with. After troubleshooting, I also wrote down the details into my notebook on what the cells should look like after each step of my experiment. This greatly reduced any doubts I had while re-running the 3rd trial today. The big lesson that I learned through all this is that trial-and-error is what research is all about and that disappointment and troubleshooting are also integral parts of the research progress. We should not only seek to get the data we want, but to also appreciate the lessons that we are able to learn when experiments fail.

Getting Started with Sequencing Analysis

It has been a few weeks and I thought that I would give some updates on my progress so far. I’m currently replicating methods from the paper Sex Speeds adaptation by altering the dynamics of molecular evolution (1). This paper provides a useful computational workflow for identifying mutations from sequence data. This past month I have come across a huge learning curve, using software that I have never come across before, specifically GATK (Gene Analysis Toolkit). My first research task is sequencing analysis, and given that I have very minimal experience using sequencing software, it has been quite a challenge. Nonetheless, I have enjoyed learning a lot from this experience. From deciphering and creating pipelines, troubleshooting errors and running 50+ hours commands (we have a huge dataset), I’m getting closer to having a working dataset that I will use for analysis.

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The Finale: Contamination galore!

Hello all,

This is my last post of the summer! I have really enjoyed my summer research experience here. Recently, I have had a bit of a hard time with my work. Most of my transformations are contaminated. I am not quite sure what has contaminated them–but it smells funky!

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