Abdelhamid, Ahmed: Cloning and Expression of Cellulose Synthase (CesA) in Pichia pastoris for Analyzing Cellulose Production

View video presentation

Title: Cloning and Expression of Cellulose Synthase (CesA) in Pichia pastoris for Analyzing Cellulose Production

Name: Ahmed Abdelhamid

Major: Biomedical Engineering

School affiliation: School of Engineering

Programs: Aresty – RA Program

Other contributors: Akshay Thakur, Dharanidaran Jayachandran & Dr. Mohammad Irfan, Dr. Shishir Chundawat

Abstract: The main area of focus in this project is to clone and transform specific inserts of DNA eventually into yeast cells in order to collect their proteins and examine their potential production of polysaccharides. In general, cellular production of polysaccharides from sugar molecules is critical in many metabolic processes to ensure survival of living organisms such as plants, bacteria and even humans. Thus, cell wall polysaccharide synthesis is particularly of interest for biomass and bioenergy applications. There are three main objectives to complete the focus of the project critical for the next step in further research. Firstly, sequence and ligation independent cloning (SLIC) is performed for fusing cellulose synthase (CesA8 from Poplar plant) with fluorescence proteins (Skylan-S and moxMaple3) and is transformed into E.cloni competent cells. This construct is then cloned into a species of yeast named Pichia pastoris for expression. Lastly, the expressed protein is purified using affinity chromatography to get the proteins responsible for polysaccharide synthesis. In terms of further research, the next step is to image these proteins through a proposed multimodal single-molecule optical imaging instrument to study the key aspects of in planta polysaccharide synthesis and their turnover dynamics. The constructs developed here will act as model systems to study the in planta polysaccharide synthesis.