Sp'23: Week 16 (5/8/23 - 5/12/23): Wrapping Up the Semester

This week was the final chance to hope that transformation occurred, but unfortunately, there was contamination again (whose common occurrence among my projects was finally likely discovered as I had somehow formed the habit of using non-sterile microcentrifuge tubes for plasmid extractions). Three out of six of my transformation plates (which included the -Cm plate "3.24C3" and two of my +Cm plates "4.5B1" and "4.5B2") showed visible signs of contamination: Deinococcus aquaticus should show up as a plate streaked with pink, but there were white colonies of an unknown organism growing on them instead (which Dr. Tuohy assumed might be Staphylococcus-- a common bacteria found on our skin!).



Because of this, I decided to conduct a full investigation via gram stain analysis to attempt to find the source of the culprit (this was before we realized that I was using non-sterile microcentrifuge tubes). I first did gram stains of the six plates (-Cm plates "3.24C1," "3.24C2," and "3.24C3," and the +Cm plates "4.5B1," "4.5B2," and "4.5B3") and placed them onto slides "A1," "A2," "A3," "B1," "B2," and "B3" respectively, which resulting gram stains shown below (all 6 plates used for Day 2 of transformation were unfortunately contaminated): 

 



Because of this, I decided to go back a step further and run a gram stain on all 6 tubes that had been used for Day 1 of transformation, all of which had competent Deinococcus aquaticus cells placed into TGY 3:3:1, but only 4 of which had the pRad1 plasmid added. I used tube "5.4A1" (-pRad1), "5.4A2" (+pRad1), "5.4A3," (+pRad1), "5.4B1" (-pRad1), "5.4B2," (+pRad1), and "5.4B3" (+pRad1) to create gram stain slides labeled "A1-," "A2-," "A3-," "B1-," "B2-," and "B3-" respectively. Although there was a bit of color inconsistency (Deinococcus aquaticus should be gram negative / show up as pink when gram stained), the overall shape seemed to be consistent with D. aquaticus (with confirmation from Dr. Tuohy and a few lab mates to double-check). Although it might be safe to say that Transformation Day 1 was done using proper asceptic technique, I believe that it is possible the Day 1 Transformation cultures were still contaminated as the pRad1 plasmid used for transformation was not extracted into sterile microcentrifuge tubes (which could account for the variation in gram stain color). 




Because there seemed to be possible contamination in each tube from Transformation Day 1, I took a step back even further and did gram stains on 5 more tubes: 
  • "3.24A" (LB + Amp [50 ug/mL] --> slide "C1" 
  • "4.27A" (LB + Amp [50 ug/mL] w/E. coli + pRad1 from plate 4.24B6, used for extraction) --> slide "C2" 
  • "4.27B" (LB + Amp [50 ug/mL] w/E. coli + pRad1 from plate 4.24B7, used for extraction and Transformation) --> slide "C3" 
  • "5.1C1" (LB + Amp [50 ug/mL] w/E. coli + pRad1 from plate 4.24B6) --> slide "C4" 
  • "5.1C2" (LB + Amp [50 ug/mL] w/E. coli + pRad1 from plate 4.24B7) --> slide "C5" 



 Finally, as it seemed that almost all of the above were contaminated, I decided to go back to my original sources (plates) and do gram stains on them. 
  • Plate "3.24C6" (TGY + D.aquaticus) --> Slide "D1" 
  • Plate "4.24B6" (LB + Amp [50 ug/mL] w/E.coli + pRad1 from freezeback) --> Slide "D2"
  • Plate "4.24B7" (LB + Amp [50 ug/mL] w/E.coli + pRad1 from freezeback) --> Slide "D3"

Fortunately, my TGY 3:3:1 plate ("3.24C6") that held D. aquaticus did not seem to be contaminated. Unfortunately, my two LB + Ampicillin plates that held E. coli with pRad1 showed slight signs of contamination, which leads me to conclude that this may be the original source of contamination altogether. 

After all of this, I have come to the conclusion that I need to create new batches of E.coli with pRad1, LB broth, TGY broth, LB plates (with and without Ampicillin [50 ug/mL]), and TGY 3:3:1 plates (with and without Chloramphenicol [3 ug/mL] from scratch. With this, I finished the semester by consolidating my plasmids and figuring out which ones I will be working with during the summer (the ones marked in green have either pRadZ1 or pRad1 plasmid, and one of my next tasks will be to figure out which may be contaminated). Aside from that, that's a wrap! Cheers to another end of a fun, educational research adventure! 

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