Sonorensis Characterization Tests

 Intro

As the ANAS meeting approaches, the AMC project is largely taking the backseat, and we pivoted towards the sonorensis characterization tests this week. Much of the time this week was spent making various medias needed for the tests, and then giving them their first run. There was a run of RNA isolation done on e. coli at the very end of the week.

Methods

Skim milk media was made using the recipe on the bottle (100g/L skim milk powder + 15g/L of agar) diluted down to 100mL

SIM media was made using the recipe given on the bottle (30g/L SIM media powder + 15g/L agar) diluted to 100mL

Urease plated were inoculated with sonorensis, as well as a positive and negative control (staph. epi and bacillus respectively)

E coli UV exposure and RNA isolation as follows:

  1. Take 100μl of sample and expose to 700mJ/M^2 of UV radiation for treatment period (2 min& 4 min) 

  2. After exposure take the 100μl and add it to 400μl of TGY

  3. Incubate at 30°C for 30 minutes before moving on to RNA isolation 

RNA isolation

  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 10 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. transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through

  7. add 400μl RNA wash buffer to the column and centrifuge for one minute, then discard the flow through

  8. prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube) and add 80μl directly into column matrix and incubate at room temperature (20-30°C) for 15 minutes, then proceed with purification

  9. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through

  10. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through

  11. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through

  12. add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through

  13. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water directly to column matrix and centrifuge


Results & Discussion
Skim milk media curdled and needed to be remade. There was a possible issue with how long it was autoclaved for, or it could be an issue with the media itself. As it is milk based there is a high likelihood of the issue lying with the autoclave specifications that we originally did not give much thought to. The urease plates that were inoculated showed no results for anything, including the positive control group. Again, it is entirely possible, and likely, that this was an issue in the making of the media and will be redone with a more discerning eye and careful hand. RNA isolation was completed, but needed a cleanup. As spring break is next week, it was decided to just do a completely different run when we return, as keeping our sample on ice for an entire week is definitely less than ideal.

Conclusion
The plan as we return from spring break is to continue giving most of our focus to the sonorensis characterization tests. Another round of RNA isolation is planned, though it is largely on the back burner for now. The SIM test will need to be redone, the skim milk media needs to be remade, and the urease plates need to be done again as well. The plates for sporulation were left out over the week and will be checked once we return from spring break.

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