Novel imaging approaches to track the movement of proteins and small molecules within bacterial cells and biofilms

• Main Author: Kumaravel Kandaswamy
• Other Authors: Kimberly Kline
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Engineering::Chemical engineering
• Online Access: http://hdl.handle.net/10356/72495
• Institution: Nanyang Technological University

Bacterial cells often exist in the form of sessile aggregates known as biofilms. Such biofilms are capable of producing adhesive Extracellular Polymeric Substances (EPS) or matrix. The biofilm matrix is often composed of biopolymers such as polysaccharide, proteins, and extracellular DNA (eDNA). Additionally, biofilms also contains phage particles that play a protective role during early stages of biofilm cycle. The matrix of the biofilm is also associated with establishment of chemical gradients, which contributes significantly to the physiological heterogeneity of the biofilm. The aim of this research was to demonstrate the use of fluorescence microscopy techniques to better understand and quantify the spatial organization of proteins and polysaccharides in single cell (Enterococcus faecalis) and biofilm (Pseudomonas aeruginosa) model systems, respectively. In doing so, high-throughput immunofluorescence assay was used to quantify the phenotypic variation in protein localization patterns in hundreds of E. faecalis cells. Furthermore, using Fluorescence Correlation Spectroscopy (FCS) the absolute diffusion coefficients of dextran molecules and phage particles entering P. aeruginosa biofilms was estimated. In essence, this research will provide technical insights on applicability of imaging tools to quantify the process occurring in single cell and biofilm model systems.