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.

中文翻译：

---

无细胞核糖体组装和蛋白质合成系统的高通量优化周期

建立变体核糖体提供了揭示核糖体生物发生基础的基本原理以及制造具有改变特性的核糖体的机会。但是，细胞活力限制了可以对核糖体进行的突变。为了解决这个限制，最近开发了一种自下而上的体外整合合成，组装和翻译（iSAT）方法，用于构建核糖体。不幸的是，iSAT复杂，昂贵且对研究人员费力，部分原因是包含20多种成分的反应缓冲液的成本很高。在这项研究中，我们开发了大肠杆菌中的iSATBL21Rosetta2细胞裂解物，一种常用的细菌菌株，具有成本效益高的基于多糖和核苷酸单磷酸的代谢方案。通过实验方法的进化设计，我们筛选出490个反应条件以优化反应缓冲液，从而使蛋白质产量比基本情况提高了10倍。本文介绍的计算指导，无细胞，高通量技术增强了我们进行多组分合成生物学项目的方法，并为重新利用核糖体做出了努力。