(353d) Towards the Construction of Synthetic Ribosomes in Vitro

In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route towards building minimal cells for synthetic biology, and permit the construction of ribosomal variants with new functions. Towards these long-term goals, we are developing an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we discuss recent improvements to the iSAT technology.  Specifically, we describe the impact of transcriptional tuning and physicochemical optimization on iSAT activity.  For example, we increased transcriptional efficiency through synthetic 3’ modifications to the rRNA gene sequences and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Collectively, our systems modifications have enabled more than 1000-fold increase in iSAT actitvity over the last 3 years. This advance will enable future studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. Further, it sets the stage for purely in vitro ribosome synthesis, the fundamental breakthrough needed for building a synthetic chemical system capable of self-replication and evolution from small molecule building blocks.