Week 3 (9/19/22 – 9/23/22): Back to the Drawing Board

 

This was the week where after running transformation and plating on Friday (a third attempt), we were hoping that we would have transformed Deinococcus radiodurans such that it would have shown antibiotic resistance via integration of the pRADZ3 gene into its own genome. However, this unfortunately didn’t happen, for reasons that we are unsure of (so far). When we checked our plates on Day #4, 5, there was still no growth, indicating no transformation.

Because of this, it is time to go back to the drawing board and are looking over every piece of the transformation protocol, from inoculation of E. coli (with its necessary plasmid that will be extracted for transformation) and  inoculation of D. rad (which is the species of Deinococcus that will be used for transformation) to plating of the hopefully transformed bacteria.  

Now, an interesting factor to consider while looking at steps to transformation [on paper] for a third time  is to consider a factor that may make the difference in transformation procedures, so much so that it may be necessary to adjust the previously-provided procedures. What is interesting to note is that when our colleague did successfully attempt transformation of Deinococcus radiodurans with a plasmid, the plasmid utilized was smaller than either of the plasmids used by us. So even though our transformation process has yet to work out, we do have a new way we will be going about it this next time:

Our next part of the transformation project will include running three transformations at once, with each transformation using a different plasmid: pRADZ, pRADZ1, and pRADZ3. pRADZ is the smaller plasmid used by our colleague through which he successfully attempted transformation. Now, as we run our experiment again, if we are able to successfully obtain transformation with the smaller plasmid (pRADZ) and not with either of the larger two plasmids (pRADZ1 and pRADZ3), that will mean two successes. First, it will mean that we actually successfully transformed a bacteria for our first time. Second, it can signify that we have been following the provided protocol properly, but may need to adjust some of the conditions or steps taken throughout some part of the transformation process (perhaps allowing more time than 45 minutes in the incubator while agitating every 15 minutes, or perhaps by changing the minimum concentration of plasmid needed for transformation.

It all is an interesting process, and after having mapped out as many steps as possible for transformation, come next week we should be able to begin steps toward transformation once more!



Comments

  1. EricaSeptember 30, 2022 at 5:39 AM

    Marisa, nice work this week despite the unfortunate result. Better luck on your next transformation!

    ReplyDelete

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