Week 3:
Carver College of Medicine

June 9, 2014

This week I attended the Survival Skills Workshop for Young Researchers held by the Biosciences Program of Carver College of Medicine for all undergraduates doing summer research in UI. Four professors from the medical school plus the Director of Admissions gave us presentations on how to keep a lab notebook, give oral and poster presentations, write personal statement and research statements for graduate school applications, and the admission procedures of UI. I also met and talked to many science majors from all over the country. UI also provides undergraduates with weekly seminars given by UI professors with topics challenging but still appropriate for undergraduate science majors. The first one of this seminar series began this week and it was about using Drosophila, known as the fruit fly, to study human diseases.

There are many different kinds of luciferase assays that we need to do for our project, and my first set of luciferase assays began this week. Luciferase is a very sensitive genetic reporter due to the absence of endogenous luciferase activity in mammalian cells. Luciferase catalyzes ATP-dependent D-luciferin oxidation to oxyluciferin, producing light emission at 560 nm, which was what I was going to measure using the luminometer. The light emitted from the reaction was also directly proportional to the number of luciferase enzyme molecules, which were secreted by transfected neurons only when they were alive. We were working with two types of luciferase, Gaussia luciferase (Gluc) and Nano luciferase (Nluc). The subjects of this assay were three differently-transfected neurons, Mff-A (with splicing sequence of 0000), Mff-A-VPE2X, and Mff-D (with splicing sequence of 0011), which were expected to cause mitochondrial fragmentation (thus more neuron deaths), inefficient binding to Drp1 fission protein (thus less neuron deaths), and no change in neuron survival (thus used as the control). We treated these three different neurons with only the cell culture media (as the control), glutamate, and rotenone, and rotenone was expected to cause more cell deaths than glutamate, which was also neuronal inhibitive. Moreover, since we transfected some neurons that expressed both Gluc and Nluc, I also needed to make sure that the two luciferase would only bind to their respective substrates and one assay buffer would also work for the other enzyme.

I also did midiprep this week, which was used to extract and purify plasmid DNA from the cells (usually E. coli). Plasmid preparation involves three steps: growth of bacterial culture, harvesting and lysis of the cells, and purification of plasmid DNA. Based on the size of cell cultures and the desired plasmid yield, similar but different procedures should be used for plasmid DNA collection, the most common of which are miniprep and midiprep. First, the cells were lysed by buffers based on the NaOH/SDS lysis method of Birnboim and Doly. After equilibration, the lysate was loaded by gravity and simultaneously cleared by the column filter. The desired plasmid DNA would bind to the silica resin. After an efficient washing step, the plasmid DNA was eluted, precipitated, and dissolved in 10 mM Tris/HCl buffer (pH 8.0 – 8.5) for further use. Finally, we used the NanoDrop spectrophotometer to measure the absorbance of the eluents at 260 nm, which was the wavelength at which DNA absorbed light. The instrument would automatically calculate the concentration of DNA based on the measured absorbance and the Beer’s Law.