High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System

Building
variant ribosomes offers opportunities to reveal fundamental
principles underlying ribosome biogenesis and to make ribosomes with
altered properties. However, cell viability limits mutations that
can be made to the ribosome. To address this limitation, the in vitro integrated synthesis, assembly and translation
(iSAT) method for ribosome construction from the bottom up was recently
developed. Unfortunately, iSAT is complex, costly, and laborious to
researchers, partially due to the high cost of reaction buffer containing
over 20 components. In this study, we develop iSAT in Escherichia coli BL21Rosetta2 cell lysates, a commonly used bacterial strain, with
a cost-effective poly sugar and nucleotide monophosphate-based metabolic
scheme. We achieved a 10-fold increase in protein yield over our base
case with an evolutionary design of experiments approach, screening
490 reaction conditions to optimize the reaction buffer. The computationally
guided, cell-free, high-throughput technology presented here augments
the way we approach multicomponent synthetic biology projects and
efforts to repurpose ribosomes.