Week of 08/19/24

 



Introduction
Transformation of Deinococcus aquaticus strain P81. The transformation of Deinococcus aquaticus has been attempted by many students in Dr. Tuohy’s Biotechnology Lab without success thus far. The P81 strain, containing certain pili genes that the reference genome lacks, was hypothesized to perhaps have a better chance to transform. Transformation, or the uptake of external DNA into a cell, is partially facilitated by pili genes thus giving us hope that this strain will transform more easily than the reference genome. 
Methods
A TGY flask of P81 is selected. Samples of P81 are aliquoted into 1ml portions contained within Eppendorf tubes. One Eppendorf tube is set aside for gram staining and for finding an OD value. After obtaining a clean gram stain and procuring an OD value near 1 (In this case the OD value was 1.32). 1 ml samples of P81 were aliquoted into three more Eppendorf tubes.  We pellet these cells. We resuspend the cells in 100 microliters of TGY broth. We add 40 microliters of 0.3M calcium chloride to each sample and mix.  We ice the samples for 15 minutes. We take 30 microliters of the sample and 10 microliters of the plasmid and mix in brand new Eppendorf tubes. The plasmid should have a concentration of 200-400 ng/ microliter for a total mass of 1 microgram per sample. PCR water is used in place of plasmid for the third tube as a control. We incubate the tubes for 90 minutes at 30 degrees Celsius while inverting every 15 minutes. We add 2 ml of TGY and incubate for 24 hours at 30 degrees Celsius. Finally, we will plate the samples on TGY plates and TGY CM plates (2 of each per sample). The TGY CM plates have a chloramphenicol concentration of 3 micrograms per milliliter that is made by mixing 17.6 microliters of stock chloramphenicol (34000 micrograms per milliliter) into 200ml of tgy broth. 

Transformation Protocol
1) 1 ml cells OD value of 1 (late log)
2) Pellet
3) Resuspend in 100 microliters of TGY
4) Add 40 microliters of 0.3 CaCl2 mix
5) Ice 15 minutes
6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA)
7) Mix
8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes.
9) Add  2 ml of TGY and Incubate for 24 hours
10) Plate cells on TGYA and TGYA CM plates
11) Use Cells mixed with PCR water as a blank

Results
Growth on all of the control plates and no growth on the chloramphenicol plates.
Discussion
The cells were not successfully transformed. The cells did not successfully take up the PRAD1 plasmid (first grown in E. coli) which would offer resistance to chloramphenicol. This resistance should have allowed the P81 strain of Deinococcus aquaticus to grow on plates infused with chloramphenicol. 

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