S-STEM Scholar Alex Hugenberg-Cox Blog

    This week we learned about microscopy as well as did RNA isolation, qPCR and inoculated a number of biochemical tests.      Chad spent one day teaching us about the florescent microscope and showing us the basic parts and procedures. We learned about the four channels that showed different colors and different parts of cells, like blue for the nucleus and green for the cytoskeleton/cell wall and red for cytosol. He showed us how to prepare the slide then went through the procedure, and showed us around the program.      This week we did the RNA isolation again.  The UV exposure was performed at  70  mJ/cm^2 for two minutes, with the  E.coli  being grown that morning then normalized, exposed and diluted. After the 30 minute recovery at 30 ° C we moved on to RNA isolation. With one round of bead beating we did the procedure like normal with one change. Instead of working out of a normal tube holder i switched to working ...

     We started this week off with the results of the inoculated multimedia test last week. W e made and inoculated TSA, NB, R2A, TGY, TGY salt at 1% 2% and 3%, Mueller Hinton, EMB, Mannitol, Maconcy, PEA and LB media. However Deinococcus sonorensis  only grew on TGY, R2A and slightly on NB. The mannitol plate had slight growth but after letting it grow an additional two days and it was eventually decided that the initial few colonies did not count for actual growth on the media.      In addition to the media from last week, we have decided to make altered TGY and R2A media to see how the bacteria would react to different levels of nutrients. A 1/2 TGY, regular TGY and x2 TGY and a  1/2 R2A, regular R2A and x2 R2A. We did have to remake some after issues with the autoclave ruined about half of the altered media, but we just remade it.     The biggest thing about the initial R2A and TGY plates in the multimedia test wasn't the fact that i...

     This week I have been joined by 5 new lab members as well as been given a second project. The project will be characterizing  Deinococcus sonorensis  as there are a number of on-going projects working on it, especially a very important bioinformatics project just on sonorensis being done by Lani. We will be looking at biochemical tests as well as some morphology ones.      The main split will be Madison and Carime working on the AMC project while Emme, Brian and Diana work on the characterization project. All of them will be kept up to date on the other projects and join when able, as well as make sure to see as many procedures as possible across both projects, but their main focus will be on one of the projects.      We started this week off with an ethanol precipitate while looking into and gathering media recipes for a multimedia test. The ethanol precipitate was performed on the six samples from last week with the hopes tha...

    This week was a short week as i was out for a few days. Most of the time spent in lab was spent helping the new people learn various skills or equations they will be using in lab. i was able to redo the RNA isolation, but overall most of the time i had in lab was spent teaching. We covered things like making media, C1/V1 equations and the nanodrop as well as things like general lab layout and where to find various necessities.      We were also able to talk more about the issues with the organic contamination and after reviewing the procedure it was realized that while the RNA isolation was normally done on Deinococcus, E.coli  did not have the same strength cell wall. While ten cycles of bead beating was necessary to break open the  Deinococcus cells, the same amount of cycles was destroying the  E.coli cells and causing the high levels of organic contamination we had seen.     The UV exposure was performed at  70  mJ/cm^2...

      This week was prepping for the first LexA E.coli  qPCR, meaning we first had to make sure that the UV limits were correct then perform an RNA isolation. The beginning of the week was spend busy with biotech bootcamp for the first few days, but after that i got started with the UV exposure to ensure that the levels that we expected to see significant impact wouldn't kill them. For this i did a simple experiment by exposing them at three different levels of UV radiance (50, 60 and 70 mJ/cm^2) for two different times (1 and 2 minutes. All of the samples were exposed then plated alongside a control, and grown till the next day. All of the plates had growth so we were able to use the highest levels and times at 70  mJ/cm^2 for two minutes during our RNA isolation.      The next day I did the RNA isolation, diluting the samples grown that morning (due to the doubling time of E.coli) to an OD of 1 +/- .05, then exposing 100 μl and after letting the...

     This semester I am still continuing work with the AMC (activated methyl cycle). This project has been continued from the 2023 spring semester, through the summer and fall semesters. While the experiment has changed throughout the semesters the core of the project has remained the same: looking at the gene expression change after oxidative stress in a species within the Deinococcus genus.       This semester I am taking a step back from the main AMC project, and instead focusing on a different gene (metK) within the cycle. While this is still very similar to the main project is covers a different gene and does not go The goal of this project is to develop a verified procedure using E.coli , see metK's response to UV exposure within Deinococcus caeni , and have a poster for this seasons conferences.  The reason we are looking into E.coli at the beginning instead of going straight into  D.caeni is so that we can verify/validate our qPCR te...

      This week we went back to our own oxidative stress experiment. After a long discussion about our lack of results when using Hydrogen peroxide as a stressor, we decided to switch our stressor from that to Sodium Hypochlorite (NaOCl). Even though we never got to test our results from the exposure where we introduced catalase, Chad was certain they would yield results no different than any of the tests we had done before. Instead it was decided to go with Sodium  Hypochlorite, from bleach.      Before we could start our actual experiment, we would need to see what percentage of sodium hypochlorite would be ideal. We would be using agar plates, and mixing the cells with different dilutions of NaOCl and inoculating. The plates would than incubate for 48 hours before establishing how each dilution percentage effected the cells. We started by doing a dilution from household bleach at roughly 8% sodium hypochlorite, diluting down...