Experimental and Biophysical Modeling of Transcription and Translation Dynamics in Bacterial- and Mammalian-based Cell-Free Expression Systems

Abstract

Cell-free expression (CFE) systems have been used extensively in system and synthetic biology as a promising platform for manufacturing proteins and chemicals. Currently, the most widely used CFE system is in vitro protein transcription and translation platform. As the rapidly increased applications and uses, it is crucial to have a standard biophysical model for quantitative studies of gene circuits, which will provide a fundamental understanding of basic working mechanisms of CFE systems. Current modeling approaches mainly focus on the characterization of E. coli-based CFE systems, a computational model that can be utilized to both bacterial- and mammalianbased CFE has not been investigated. Here, we developed a simple ODE (ordinary differential equation)-based biophysical model to simulate transcription and translation dynamics for both bacterial- and mammalian-based CFE systems. The key parameters were estimated and adjusted based on experimental results. We next tested four gene circuits to characterize kinetic dynamics of transcription and translation in E. coli- and HeLa-based CFE systems. The real-time transcription and translation were monitored using Broccoli aptamer, double stranded locked nucleic acid (dsLNA) probe and fluorescent protein. We demonstrated the difference of kinetic dynamics for transcription and translation in both systems, which will provide valuable information for quantitative genomic and proteomic studies. This simple biophysical model and the experimental data for both E. coli- and HeLa-based CFE will be useful for researchers that are interested in genetic engineering and CFE bio-manufacturing.