Development of in vitro ribosome assembly system for ribosome engineering with ribosome biogenesis factors YrdC and SrmB

Ribosomes, the ubiquitous macromolecular machines responsible for protein synthesis, are required for cellular activity. This work, conducted with meticulous attention to detail, aimed to develop and optimize approaches for in vitro ribosome synthesis and assembly to establish a ribosome engineering platform. The study focused on several critical aspects, including optimizing ribosome and S150 extract separation, developing quantitative fluorescence-based assays for ribosomal activity, isolating and characterizing total proteins (TP70) and total RNA (TR70) from 70S ribosomes, evaluating in vitro transcription methods for rRNA, and investigating the integrated synthesis, assembly, and translation (iSAT) system. The study was effective in isolating highly active ribosomes and S150 extracts, as well as developing a quantitative fluorescence-based ribosome activity assay. This assay, a significant development, provides a new tool for researchers in the field. Optimized procedures for TP70 and TR70 isolation were created, and SDS-PAGE analysis confirmed the existence of essential components. This study lays the groundwork for future research in ribosome engineering and opens up exciting possibilities in antibiotic discovery, non-natural amino acid incorporation, and protein production optimization for biotechnology. The potential impact of this research on future studies is inspiring and motivating.