Week 4 (9/26/22 – 9/30/22): The Great Preparation

Since transformation of our Deinococcus radiodurans fell through, this week's focus was on finalizing the process to this time run three sets of transformations (including the creation of all broths we will need for inoculations, nanodrop readings, plating, and more). So far, we have tried running transformation three separate times (each time becoming a bit faster and a bit more precise in our methods), but since we didn't achieve transformation in each one, we decided to take a step back to try and see if we could try something different. 

Our classmate had previously been able to run transformation successfully with a plasmid smaller. So to make sure that we actually are following the correct protocols, we will be running three transformations with three separate plasmids (and using Deinococcus radiodurans), one of which will be the smaller plasmid used by our classmate to perform his own transformation. If we are able to achieve transformation at all (especially if we can do it with this smaller plasmid), then we'll know that we have been following the protocol correctly, but that we may need to modify one or some of the factors affecting the transformation process (such as the amount of time that the bacteria are incubated during the incubation/agitation process, or perhaps even by changing some of the conditions of cell competency before undergoing the transformation process). 

Below is a picture that shows the three different plasmids that we will be transforming, as well as some of the planning process from start to finish (including how much material will be needed per step, how long it'll take to do each step, which steps overlap, what type of help we will need by others, and more). The entire process is quite long and intricate, but between the experience of running transformation multiple times and planning for our next steps. I have learned quite a bit about how to improve my own methods. 

With this, since one of the very first steps in the process is to make sure you have enough broth for inoculations (LB broth & TGY broth), and for the plates, I went ahead and made the following broths: 

  • LB (used for inoculating the E. coli with the plasmid in Ampicillin and for baseline measurements with the nanodrop) 
  • LB + Agar (used for inoculating/plating E. coli with the plasmid in Ampicillin) 
  • TGY (used for inoculating D. radiodurans and for baseline measurements with the nanodrop) 
  • TGY + Agar (used for inoculating/plating D. radiodurans with and without Chloramphenicol 
We were able to have all of our broths autoclaved. Because of this, we will be ready to move onto the next steps, including the creation of competent cells this upcoming week. 

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