Cell-free protein synthesis (CFPS) is often touted as the future of protein production for functional studies. This thesis focuses on how altering the amino acid concentration affects the synthesis of proteins in PUREfrex 2.1, a commercial recombinant CFPS system. These experiments will inform the optimization of a custom recombinant CFPS kit for membrane proteins (MPs), which is currently under development in the Daniel lab. The protein targets analyzed were soluble super folder green fluorescent protein (sfGFP) and MscL-mEGFP, a fusion protein of the membrane protein MscL and a mEGFP folding reporter. The experiments focused on determining how doubling the concentration of select groups of amino acids affects the synthesis of a soluble and MP target in PUREfrex 2.1. The amino acid mixture included with PUREfrex 2.1 has an equimolar concentration of the twenty common amino acids.1,2 Additional amino acids were supplemented into the reaction mixture to determine the effect of increasing the concentrations of several amino acid groups on protein synthesis relative to the equimolar case. The sfGFP and MscL-mEGFP synthesis reactions were run using the equimolar mix in addition to mixes where the concentrations of negative, positive, polar (noncharged), aromatic, and aliphatic amino acids were doubled. sfGFP was the soluble protein target. Increasing the concentration of every amino acid mixture led to increases in average total and fractional sfGFP yield, but only doubling the concentration of negatively charged amino acids led to a statistically significant increase. Doubling the concentration of negatively charged amino acids (aspartate and glutamate) led to a ~23% increase in total yield and a ~9% increase in fractional sfGFP yield relative to the equimolar case. After analyzing the effect of amino acid concentration on soluble sfGFP, the next target was a membrane-bound target, MscL-mEGFP. For this target, I analyzed the effect of using liposomes as a membrane mimetic in addition to altering amino acid concentrations. Adding liposomes to MscL-mEGFP synthesis reactions run using the equimolar amino acid mixture led to a ~200% increase in total yield and ~120% relative increase in fractional yield, both of which were statistically significant increases. Adding DOPC liposomes and doubling the concentration of aromatic compounds led to a ~20% increase in total and fractional MscL-mEGFP yield compared to the equimolar case with DOPC liposomes. Despite statistically significant increases in MscL-mEGFP yield with the addition of liposomes and increases in the concentration of aromatic amino acids, the fractional yield of sfGFP (~37-45%) is still ~17-20 times higher than MscL-mEGFP yield (2.61%) in PUREfrex 2.1 depending on the amino acid mixture used. The gap in fractional yield between soluble and membrane protein targets indicates the need for recombinant CFPS kits optimized for synthesizing MPs. Future work will focus on analyzing whether these synthesis trends are universally applicable to groups of proteins or specific to individual proteins.