CADGE 2.0, Transcription-Translation-Coupled DNA Replication is Improved in a Chemically Modified Cell-Free System

Abstract

In vitro directed evolution in synthetic microcompartments can generally support the evolution of genes with functions beyond affinity. The main challenge in the implementation of 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 which 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 that allows the clonal amplification of linear gene-encoding DNA and coupled, in situ transcription-translation of the gene of interest. Here, we show that clonal amplification is highly sensitive to the cell-free systems composition and that robust, highly efficient cell-free DNA amplification via the CADGE platform can be achieved by replacing standard vendor-supplied energy mixes with DNA replication-optimized, homemade counterparts.