Festival of Science 2017 | St. Lawrence University Biochemistry

Festival of Science 2017

Festival of Science was held April 28th, 2017

Vanessa Chilunda "Effects of Growth Stages and Sub-inhibitory Antibiotic Concentrations on Amyloid Protein Formation by Soil Bacteria"
Biochemistry Faculty Advisor: Nadia Marano

Functional amyloids are an important component of biofilms formed by bacterial communities. These amyloids are important due to their hydrophobic nature and other biochemical properties that help bacteria resist harsh conditions such as heat, pH, hosts’ immune systems and even some antibiotics. Studies have shown that subinhibitory antibiotic levels serve as signaling molecules that increase biofilm production and consequently bacterial resistance properties. Thus, I decided to study the effects of subinhibitory tetracycline concentrations on amyloid production in biofilms. Thioflavin T (ThT) spectroscopic assay was used to quantify the amyloid production. I found a statistically significant increase in amyloid production in one of the isolates when treated with 0.2 μg/ml and 0.5 μg/ml tetracycline concentrations. However, at higher tetracycline concentrations, amyloid production decreased. In order to elaborate on these results, I investigated the time taken by the bacteria isolates to produce maximum amounts of amyloid proteins in their biofilms. Bacteria concentration was estimated using absorbance at a wavelength of 600nm on spectrophotometer, and compared to bacteria count under a phase contrast microscope. I found that there was maximum amyloid production around day five when bacteria were cultured in liquid media. However, growing the isolates in liquid media for a longer period of time resulted to less cell-associated amyloid fibers compared to when cultured on agar plates. Currently, I am measuring amyloid production by bacteria isolates grown on agar plates instead of liquid media with different sub-inhibitory tetracycline concentration at different growth periods.

Kirsten Padalis  "Optimizing a Thioflavin-T Assay using the Microplate Reader"
Biochemistry Faculty Advisor: Nadia Marano

Amyloids are proteins that are characterized by fibrils that have beta sheets which are perpendicular to the fiber axis. There are functional amyloid fibers that can be produced by bacteria as part of their biofilms. An assay optimized by Jordan Koloski using Thioflavin T, adye that fluoresces when bound to amyloid fibrils, was used to measure the growth of amyloids of different tetracycline resistant soil bacteria. This assay has been done by measuring the fluorescence using the Horiba FluoroMax Spectrofluorimeter. I created a new assay to be able to read and analyze the fluorescence on the Synergy HTX Multi-Mode Microplate reader. This plate reader can measure fluorescence on a 96 well plate so that more samples can be analyzed at once, which will help cut down time for gathering data allowing more trials to be performed. The assay was first optimized using aggregated insulin, which gives consistently high fluorescence then further studied with bacteria grown on plates. I concluded that the plate reader is not as sensitive at reading fluorescence as the fluorimeter. Therefore, the plate reader is a good way to check a lot of samples at once and to see which ones should be studied further using the fluorimeter.