Cell-free protein synthesis (CFPS) has emerged as an enabling biotechnology for research and biomanufacturing as it allows for the production of protein without the need for a living cell. Applications of CFPS include the construction of libraries for functional genomics and structural biology, the production of personalized medicine, and the expression of virus-like particles. The absence of a cell wall provides an open platform for direct manipulation of the reaction conditions and biological machinery. This project focuses on adapting the CFPS biotechnology to the classroom, making a hands-on bioengineering approach to learning protein synthesis accessible to students grades K-16 through developing an affordable ‘protein synthesis classroom kit’. To address the barrier of cost to entering the classroom, CFPS reaction was reformulated with the goal to reduce costs while optimizing protein yield. An inexpensive sugar was added to the reaction in varying concentrations for its potential to recycle inorganic phosphate and regenerate ATP. Phosphoenolpyruvate (PEP), an expensive energy source, was replaced with a lower concentration of 3-phosphoglyceric acid (3-PGA). We determined that adding the sugar within the range of 10-30mM did not have a significant effect on high-performing cell extracts grown in 2xYTPG for the PEP energy system, and had a slight boost to protein yield at a concentration of 10mM for cell extract grown in 2xYTP media. Although the 3-PGA system yielded less protein than the PEP system, the sugar combined with 3-PGA contributed greater percentage gains for cell extract grown in both media when compared to controls. Future work may include whether the sugar and 3-PGA worked in tandem or independently. Understanding gained from these experiments would contribute to optimizing protein yield and reduce the cost barrier for classroom use.


Bacteria | Biochemistry | Biotechnology | Chemistry | Higher Education | Laboratory and Basic Science Research | Microbiology | Molecular Biology | Science and Mathematics Education | Secondary Education


Javin Oza

Lab site

California Polytechnic State University (Cal Poly SLO)

Funding Acknowledgement

The 2018 STEM Teacher and Researcher Program and this project have been made possible through support from Chevron (www.chevron.com), the National Marine Sanctuary Foundation (www.marinesanctuary.org), the National Science Foundation through the Robert Noyce Program under Grant #1836335 and 1340110, the California State University Office of the Chancellor, and California Polytechnic State University in partnership with the Department of Chemistry and Biochemistry at the Center for Applications in Biotechnology, San Luis Obispo. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funders.



URL: https://digitalcommons.calpoly.edu/star/515


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