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

• DOI: 10.1021/acssynbio.8b00276
• Publication Date: 2018-10-24T22:15:40Z
• Authors: Filippo Caschera; Ashty S. Karim; Gianluca Gazzola; Anne E. d’Aquino; Norman H. Packard; Michael C. Jewett
• Keywords: Synthetic Biology; ribosome biogenesis; Cell-free protein synthesis

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