Building Autonomy into a Cell-Free Protein Producing Gel: Integrating T7 RNA Polymerase

Abstract

Cell-free protein production is a new technology that offers many advantages over conventional cell-based expression techniques, though low yields (in the range of µg/ml) have been a major drawback. With the advent of the p-gel system, a DNA hydrogel consisting of genes as part of the gel scaffolding, yields in the mg/ml range have been demonstrated. As p-gel expression relies on exogenous transcriptional and translational enzymes, cloning these proteins into the p-gel system would increase its autonomy. Here, we attempt to clone T7 RNA polymerase into a vector compatible with the p-gel system. Primers were designed to amplify T7 RNA polymerase from E. coli strain BL21 (DE3) as well as attach NcoI and SmaI restriction enzyme sites via PCR. The amplicon was digested and ligated with Roche’s pIVEX2.4d, a plasmid that supports cell-free protein expression in E. coli lysate with bacteriophage T7 RNA polymerase. The plasmid was then used to transform chemically competent E. coli cells. Colonies carrying the plasmid of interest were selected for via colony PCR and submitted for sequencing. Unfortunately, our samples displayed an abundance of mutations that would likely affect T7 RNA polymerase’s activity. Nonetheless, with additional work, the plasmid samples produced could be useful in p-gel expression studies.