Protein-Primed DNA Replication is Restored in a Modified Cell-Free Protein Synthesis System

In vitro directed evolution in synthetic microcompartments can support the evolution of genes with functions beyond affinity. The main challenge in implementing this strategy is the need to incorporate no more than a single DNA template molecule per microcompartment, thereby establishing a robust genotype-phenotype linkage, but that results in slow, inconsistent in vitro transcription and translation (IVTT) and poor DNA recovery after selection or screening. To address this challenge, we previously developed CADGE (Clonal Amplification-enhanceD Gene Expression), a strategy implemented in PURE (Protein synthesis Using Recombinant Elements) systems that allows the clonal amplification of linear gene-encoding DNA and coupled, in situ transcription-translation of the gene of interest. However, we found that current commercial PURE systems support substantially lower DNA replication than previously reported. Here, we restore DNA amplification in CADGE to previously reported levels by replacing vendor-supplied energy mixes with homemade counterparts previously shown to enhance rolling circle amplification.