Christopher Werner, Brigham Young University
Engineering
Preparation of DNA linear expression templates (LET) via Polymerase Chain Reaction (PCR) is significantly faster than procurement of DNA via cell growth and plasmid purification. Unfortunately, linear templates have not consistently achieved protein yields comparable to plasmids in cell-free protein synthesis (CFPS). Possible reasons for lower LET yields were investigated by producing a number of different extracts. Extracts were differentiated by varying harvest time after induction and modifying the extract preparation procedure. These extracts were tested with py71 sfGFP plasmid (producing a reporter protein) and LET’s created through PCR from the same plasmid. Protein yields obtained by fluorescence measurement were plotted against combined tRNA and rRNA amounts obtained through DNA electrophoreses and densitometry. A correlation was seen between tRNA and rRNA amounts and a normalized LET yield (LET yield divided by the plasmid yield under identical conditions). We considered two reasons for this correlation. First, the increased tRNA and rRNA indicated and increase in the concentration of cell translation machinery present, which increased the kinetics of the reaction, allowing LET’s to produce protein quicker before degradation by Deoxyribonucleases (DNAse). Second, the increased tRNA and rRNA amounts acted as a shield for mRNA from Ribonucleases, allowing more of the mRNA to be translated before LET’s were degraded by DNAse’s. Further work must be done to verify the accuracy of this correlation, as well as to determine the cause for increased LET yields in extracts with higher tRNA and rRNA amounts.