Eric Uses qPCR to Test His Samples

Con mis amigas

Written by Eric Scheider

Last week I started a new set of experiments aimed at fixing some problems we have encountered in past experiments. For the ysaDCB gene cluster, the inducing molecule nisin is the same bacteriocin we are using to disrupt its normal function. This means that it is exceedingly difficult to make sure that the natural wild type nisin is the nisin that induces the gene function and the mutant nisin is the one to disrupt cell wall function. Since the objective of this experiment is to determine the both the inducing and antimicrobial activities of nisin, we cannot use both wild type nisin and mutant nisin on our Lactococcus lactis cell cultures. To then directly compare the inducing and disrupting activity of different mutants of nisin, I need to run a fluorescence assay on the cell cultures when exposed to different concentrations of nisin. However, natural L. lactis cells do not glow so I use a modified version where the promoter of the ysaDCB gene is upstream of a gene, mCherry, that is fluorescent when activated. This requires careful measurement of small volumes and sterile conditions when loading the microtiter plate to be read by the very sensitive qPCR (quantitative Polymerase Chain Reaction) machine which can provide me with real time fluorescence data very quickly. The initial tests with this procedure showed promising results, so I will be comparing all 27 variants of nisin we have versus the wild type nisin in the coming days. Fluorescence data from these experiments will then give insight into the inducing activity of the nisin variants.
Results of IC50 assay
El glamour de una máquina qPCR
Last week I also said goodbye to some of the friends I’ve been lucky enough to make while here at IPLA. They had finished their work here in Villaviciosa so we met in Oviedo to have the best Asturian food I think I will ever have. Cachopo with a plate of fried vegetables and fruits and a rack of something similar to ribs. To drink they gave a pitcher of a mixture of cider and sangria. It was a dinner I will never forget and the night out that followed will be difficult to as well. I’m going to miss this place more than any other, I think. Home in North Carolina has been hard to miss because I’ve always known I would be back there before I knew it. But my next time in the north of Spain? God only knows when that’ll be.