Week 2 (9/12/22 – 9/16/22): Transformation – Take Two!

 This week, our major focus was on ensuring we understood every part of our transformation protocol (especially in regard to our controls) so that we could ensure a [hopefully] successful transformation! Now, one important concept that our mentor Jonathan helped us to understand was the importance of proving the viability of our competent cells (taken from freeze back), which would help to show that our competent bacteria weren’t the problem if we had no growth after transformation, and proving the stability of our environment, which would help to show that the plates held a solid environment for D. rad to grow in. Because transformed cells need to be plated after 16 hours, and because an entire weekend had passed since we transformed our cells with the pRADZ1 and pRADZ3 plasmids (in separate groups of course), we would need to restart part of the transformation process (after ensuring that we fully understood each step in our transformation process of course). 

We did this first by inoculating our already-made competent D. rad cells (obtained from freeze back) onto two different TGY 3:3:1 agar plates -- one with a minimum inhibitory concentration (“mic”) of 3 ug/mL of Chloramphenicol, and one without Chloramphenicol added at all (labeled “+Cm” and “-Cm” respectively). Both TGY 3:3:1 agar plates were then incubated at a temperature of 30 degrees Celsius and checked the next day. Great news: We had growth on the “-Cm” plate (which proved the viability of our competent D. rad cells and showed that the environment on which they were growing (the TGY 3:3:1 agar plates) were also suitable for growth). There was also a lack of growth on the “+Cm” plate, which was as expected as non-transformed / regular D. rad does not have Chloramphenicol resistance and would not be expected to be able to grow on a plate with a mic of 3 ug/mL of Chloramphenicol. 

Our competent D. rad’s growth on the “-Cm” plate verified to us that we had the green light to move onto transformation of D. rad (which would, in fact, be our third attempt – the first two being unsuccessful, both ultimately due to contamination). This time, we only worked on using the pRADZ3 plasmid to transform Deinococcus radiodurans to give it Chloramphenicol resistance. Since part of the transformation process calls for a wait time of 16 hours before plating the [hopefully] transformed bacteria, we will be checking the growth after this weekend (as plating was done on Friday, and because growth can take up to four days to occur for [transformed] D. rad). 

We also did an inoculation of E. coli that held the pRADZ1 plasmid, which we ended up extracting the next day after seeing that we did have growth of this particular E. coli in one of the two tubes (“Tube B”) that we had incubated the previous day. Two extractions were run of the pRADZ1 plasmid from E. coli, and the numbers obtained were as follows: 

These numbers, although not ideal, are a workable enough concentration that will allow us to run a gel (to check the size of the plasmid and ensure we are working with the correct one) and run transformation of D. rad again, this time with Z1. Hopefully we see growth (which would signify transformation) on Monday!

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