More Characterization

 Intro

My time this week was dedicated specifically to exposing deinococcus sonorensis to various levels of ultraviolet radiation and to view the impact on its ability to survive radiation based on what type of media sonorensis is grown on. Specifically, the medias of TGY and R2A were tested and observed for growth.

Methods


Visual diagram of process shown above

  • 3 biological reps each of TGY and R2A, grow sonorensis in broth of respective mediums
  • measure OD600 value for each sample, normalize to 1
  • expose 50 uL of sample to radiation (2500, 3000, 3500, 4000, 5000*100uJ/cm^2) for 5 mins
  • return exposed sample to 450uL of pure media, vortex to incorporate
  • inoculate onto plate of respective media
  • wait and observe for growth

Results & Discussion
Growth was seen on none of the R2A plates, but was present on the 3000 and 5000 TGY plates


It should also be noted that while trying to normalize the OD600 values before moving on to exposure, the sonorensis grown in R2A was significantly easier to normalize and less prone to "clumping" as it does in TGY. It is possible that when grown on media that is less dense in nutrients, the plaque that sonorensis forms is weakened, making it distribute more normally. This is opposed to TGY that is higher in nutirents, so it forms it's plaque as normal. As seen with the results from UV exposure, this plaque and higher nutrient density is possibly what allows sonorensis to survive doses of radiation at all.

Conclusion
Further testing should be done with UV exposure, to possibly find the threshold of radiation R2A allows for sonorensis to survive and grow, as well as the upper end for TGY, to see at what point will there be too much radiation and it can no longer recover.

Comments

Post a Comment

Popular posts from this blog

UV Exposure

  Intro Much of this week was dedicated to filling out our poster. What wasn't spent on poster work was spent on testing UV radiation tolerance again. The MRVP tests were done again and were again decided to be inconclusive. Method Method for UV exposure is exactly the same as last week, but R2A was exposed at levels of 1000, 1250, 1500, 2000, & 3000. TGY was exposed to 3000, 5000, 6000, 7000, 8000, & 9000. Results & Discussion Again, there was absolutely no growth on any of the R2A plates. The threshold is cleary far lower than we expected due to the media of R2A while, again on TGY, there was growth from the 3000 & 5000 plates. Conclusion Really all we have left moving forward is little details and pictures for the poster. All of the tests have been conducted at least once, but a few of them need to be redone still to see if we can verify a true positive/negative result. 
Read more

Week of 10/14/24-10/18/24

Image
Intro This week involved another round of RNA isolation, followed by purification and then an RNA gel and fluorometer check to ensure quality of the RNA before proceeding.  Methods Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes. RNA isolation procedure: 1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube 2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times 3. centrifuge the tube for one minute to pellet debris 4. transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through 5. Add an equal volume of ethanol (95-100%) and mix well 6. tra...
Read more

Ref Gene Progress

Image
Intro     As we return to lab for the fall, we pick up exactly where we left off from the summer. With the reference gene project we've switched species to Deinococcus deserti, and with that Alex has taken time in the week inbetween to become familiarized with it. As we are officially working on the new desiccation project as well, we have been given a second species to continue our work with: Deinococcus pimensis. Over the course of the week we plated samples of both species for 24 hour, 48 hour, and 7 day desiccation.  Methods On Monday 8/19/24, a recently grown sample of deserti was gram stained to ensure no contamination before being used for inoculation. We found gram negative rods with no contaminants, so the plate was used for inoculation. A different newer plate that had left to grow over the weekend was used for starting 48hrs of desiccation to extract RNA. The 24hr and 7 day deserti, as well as the 24hr, 48hr, and 7 day pimensis were all plated the followin...
Read more