The Ninth Week – From Murder to Mutation

At the beginning of the summer in my first blog, I attempted to explain my proposed project for this summer. The first part of that explanation was about testing the biological function of manganese oxidation, which translates to murdering a lot of bacteria and seeing if the ones coated in manganese oxides survive better. The second part I was unclear on, but I have spent most of this week working on that second project. If testing oxidation as protection translates to murdering bacteria, this new project translates to mutating bacteria. I have been trying to make a mutant of P. putida that has increased oxidation because of increased mnxR production on a plasmid.
 

A lot of my blog last week was about how to mutate the bacteria. What we did this week was screen these mutants for the one we want. The successful transformations were of Kati’s mutagenized pKG207 plasmid from February into wild-type and KG127(non-oxidizing) cells. Of these transformations, we choose the ones that were increased oxidizers and continued to re-streak to verify that they really were increased oxidizers and really were Pseudomonas putida. There were two especially promising strands of KG127 that had picked up a plasmid and were oxidizing a whole lot. So, we inoculated a culture of these in LB and let them grow overnight to get lots of these cells. We then purified the plasmid from these cells using a special protocol in a special QIA miniprep kit. So, basically, some magic happened, and we got the plasmid out of the cells. Then, we transformed these plasmids into wild-type P. putida, KG127, and E. coli. I realize I’ve just been calling one strain KG127, which means nothing to whoever is reading this. KG127 is a ΔmnxR mutant, which means there is a mutation in the DNA that interferes with mnxR production and the mutant cannot oxidize manganese. We have been using this mutant to test many environmental stresses to verify that a non-oxidizing mutant does not exhibit protection. Now, we are trying to get a plasmid that results in a lot of oxidation because of increased mnxR production. So we are using KG127 (ΔmnxR) so that all mnxR formed in the cell is from the plasmid DNA and not the original chromosome DNA. This way there will be no interference or uncertainty with where the oxidation is coming from.
 

So, we have the plasmid that hopefully has mutated mnxR and will cause P. putida to oxidize a lot. We transformed these plasmids into wild-type and KG127, and E. coli as a back-up. In case the plasmid was not taken up by Pseudomonas putida (because it’s more difficult for P. putida to transform) we would hopefully still have the plasmid transformed into E. coli to conjugate into Pseudomonas putida. Fortunately, all the transformations resulted in colonies, so P. putida did take up the plasmid! I struck out colonies from each transformation onto lept+GM plates to verify their oxidation phenotype and see if the plasmid is really what we want. I was very happy to see that there were colonies on the transformation plates, because I’ve seen a lot of blank plates in my past transformations. It feels kind of like Frankenstein, because I’m so happy that these bacteria have been mutated the way I want! We are going to purify the plasmid again and then take it in for sequencing to see what is causing the increased oxidation from the plasmid, and hopefully it can be targeted to one mutation that increases mnxR production.
 

I’ve also been working on another type of mutation this week: my final paper and presentation. I’m sure that sounds a little strange, but it’s very true. My practice presentation for the Tebo/Haygood lab group was on Tuesday, so I spent a lot of last week and most of Monday preparing for that. The way I created that presentation was by mutating (more commonly known as editing) my mid-term presentation. I expanded on a lot of the slides and added a lot of pictures.  The final presentation really was a monster, including over 15 slides and lasting over 10 minutes.  But it was worth it because my practice talk went very smoothly and I articulated my research very clearly. I got some good feedback afterwards and I feel like I’ve got a very solid presentation to continue to perfect next week, although I’ll definitely have to cut it down a bit.
 

My other mutant is the paper; this one was a monster to begin with though. I basically did a brain dump of my entire summer project on oxidation as a protective mechanism from environmental stresses into one 5-6 page long summary. Right now it is not organized at all, but it has a lot of information for me to sort out. My mentor is performing the first step in the mutation by reading it and beginning the editing process, letting me know what to change to make it fit a scientific research paper format. Next week it will be ready to be mutated from a monster to a coherent scientific paper.
 

I really enjoy the process of mutating (or editing if you prefer). I take great pleasure in organizing and re-working sentences and paragraphs to be as clear and simple as possible, so I’m really looking forward to finishing my paper. I also have the challenge of cutting down my presentation to attempt, which I already have a few ideas for. And we can’t forget the bacteria. The real mutant must be taken care of next week and hopefully Kati and I have successfully created the mutant she was planning to make in the beginning of the summer. I have decided this week that I prefer the process of mutating over murdering. That could just be because murdering bacteria sounds awfully mean or it could be that I am a perfectionist and I like to mutate things until they’re just right. Either way, I have a lot of mutant monsters to deal with in the upcoming week.