Date of Award

11-2019

Degree Name

MS in Biological Sciences

Department/Program

Biological Sciences

College

College of Science and Mathematics

Advisor

Christopher Kitts

Advisor Department

Biological Sciences

Advisor College

College of Science and Mathematics

Abstract

Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. Although there have been many advancements toward CFPS development, the democratization of the platform to a wide variety of educational, research, and industrial institutions has lacked due to an absence of resources for new users as well as a limited number of developments toward redesigning the tedious and time-consuming protocols to generate robust cell extract. To address these challenges to CFPS implementation, a comprehensive review spanning numerous cell lines with their respective applications, methodologies, and reaction formats were provided in addition to detailed protocols outlining the process of going from E. coli cells to a completed CFPS reaction. Together, these resources provide the scientific community with easily accessible resources for CFPS implementation. Moreover, the aforementioned protocols were redesigned from a four-day process into one that may be completed in under 24-hour’s time with very little researcher oversight. The resulting workflow maintained the robustness of prior methods but generated 400% more extract compared to traditional methods via a set-it-and-forget-it approach. To date, the works presented herein have garnered tremendous viewership from the CFPS research community with a substantial following among all three of the articles. Moving forward, I anticipate that these works will continue to bring new users into the CFPS field through the ease of access to these resources and through the advance of the simplistic and reproducible new workflow for preparation of robust E. coli cell extract.

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