Optimization of Cell‐Free Protein Synthesis Through Identification of Rate‐Limiting Factors

Abstract

We have implemented the Genetic Code kit into classrooms at Cal Poly, San Luis Obispo for a hands‐on inquiry‐based module of transcription and translation. Our Genetic Code kit is based on cell‐free protein synthesis (CFPS) which captures the transcription and translation machinery required for protein synthesis in vitro, allowing for direct manipulation of protein production. By obviating the use of a living cell, CFPS allows early‐career STEM students to observe protein synthesis. To observe the process, superfolder green fluorescent protein (sfGFP), is used as a reporter protein. The detectable fluorescence of sfGFP gives students real‐time feedback of protein synthesis occurring. However, this process takes upwards of three hours for complete synthesis and maturation. To be more applicable in an educational setting, CFPS needs to occur in a more time‐efficient manner. Thus, this work focuses on identifying the rate‐limiting factors of CFPS. These rate‐limiting factors can be organized into two classes. The first class consists of energy sources and small molecules, and the second class consists of protein‐based translation factors. Direct titrations of these small molecules and proteins into CFPS reactions were performed. Here, we report our findings of key additives that increase the translation rate in cell‐free. Additionally, miscellaneous small molecules, such as osmolytes and detergents, have also been found to increase the reaction rate. The translation factors currently being studied are: prokaryotic initiation factors, elongation factors, release factors, ribosome recycling factor and the methionyl transferase. By optimizing the process of CFPS, the platform can become time‐efficient for a single lab setting. To date we have decreased reaction time by 30%, significantly improving the broad applicability of the Genetic Code Kit in classrooms.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, the Bill and Linda Frost Fund, and NSF‐1708919.