S-STEM Scholar Kalen Mullenix's Blog

 To start the week we had put the 3-Way Insert Fragment and the PRAD 1 Vector into the Gibson master mix and let it incubate at 50C for 1 Hour. We then had to wait a couple days so that the confirmation primers were ready to use and once they were ready we put our Assembled Plasmid into two different PCR reactions. One reaction with the Chloramphenicol confirmation primer and the other with the Ampicillin conformation primer since the size difference between the primers meant that the extension time and annealing temperatures would be different. Once this PCR was done running, we had put this on a gel. With the 1st lane being the 1KB ladder, the 2nd lane was the Chloramphenicol confirmation primer (3503bp), and the 3rd lane was the Ampicillin confirmation primer (4681bp). The results initially were disappointing. We initially believed that the Gibson Plasmid did not work, however there could be a chance that in relation to all the side products the Gibson Plasmid has very low conce...

 We ran PCR on the Gibson product that we had gotten from last week. In this PCR we had used the Q5 Polymerase since it would give us more yield that the usual Taq polymerase that we use. For this PCR we had ran it on the PCR product that we had gotten from last week and on the original Gibson Assembly prior to any PCR. On top of that we ran Long Amp PCR on our PRAD 1 Plasmids again to give us more sample of PRAD 1 vector to work with when attempting Gibson Assembly on the entire plasmid. The next day we ran one of the bigger gels, the ones that have around 15 wells. We needed to use this one since we had 9 wells that we had to fill since we had 4 Gibson PCR products, 4 Long Amp PCR products, and the 1kb ladder. This gel did take a little longer due to the size difference, however once it was done we viewed it under UV light immediately. What we saw was that the PCR on the already PCR’d Gibson products appeared to completely disappear which was a little worrying to us at first. Th...

 At the start of the week we had prepared 3 plates from freezebacs. One being D. Rad, one being PRAD 1 in LB Amp, and the other being PHRAM in LB Kan. We started to make sure that these were constantly growing in case we needed to do any plasmid or genomic extractions to get new samples for the Gibson Assembly project. We had also inoculated D. Metalli into R2B and let it incubate for 24 hours to observe any biofilm formation. Surprisingly by the next day there appeared to be some biofilm on the bottom of the flask, while at this point it was still pretty weak and there was little biofilm growth, any growth was a surprise since that was not what I had hypothesized. Under gram stain the cells were consistent to growth in TGY Broth in which they were not uniform and had a stringy structure, however we did not see any biofilm so we had let it sit in the incubator for another 24 hours to see how much more would grow.  Slight Biofilm Growth of D. Metalli in R2B The next day we did ...

I feel as though it is appropriate to combine these two weeks into one blog post since during these weeks we had not done much in terms of our project in terms of procedures. During the first week we were mainly focused on getting our poster ready for the Arizona Nevada Academy of Science conference that was on 4/13. So, we were designing our poster throughout the majority of the week to make sure that our poster was as professional as possible and appropriately displayed our findings from the last few weeks that we had spent working with D. Metalli. Working on the poster and preparing for the conference took a majority of our time in the first week so we didn’t do any further procedures on D. Metalli and its ability to grow in R2A to observe for biofilm formation.  Our Poster on D. Metalli Characterization at the Arizona Nevada Academy of Science  In the second week there was not much that was done as well, somewhat due to us not really sure on how we wanted to move forward. ...

 D. Metalli is reported to form a biofilm when grown in a nutrient rich broth such as TGY, however we had yet to try this since our focus was on the biochemical tests which were all conducted on TGY and R2A plates. So, once we heard that this was a characteristic belonging to D. Metalli we had inoculated some of the bacteria into a 25mL flask of TGY and let it sit the in shaking incubator for 24-48 hours. After 48 hours of incubation in the shaking incubator there seemed to be no presence of a biofilm. There were no changes in the morphology when seen under a Gram Stain as well. I had asked one of the mentors about this and he suggested that I try leaving the flask in the incubator that doesn’t shake and leave it in there for 24 hours to see if any change occurs. After 24 hours there was a film like substance on the bottom of the flask that I had not seen before in any other species of Deinococcus.  Visible Biofilm Formation at Bottom of Flask (1) Visible Biofilm Formation at ...