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
