Abstract
Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. To that end, we have developed an integrated synthesis, assembly, and translation technology (termed iSAT) for the in vitro construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. iSAT allows for the simultaneous in vitro synthesis of ribosomal RNA (rRNA), assembly of rRNA with purified ribosomal proteins, and transcription and translation of a reporter protein as a measure of ribosome activity. Here, we describe the development of, and recent improvements to, the iSAT system. Key breakthroughs include refining extract preparation, tuning the transcriptional balance of rRNA and mRNA production, and adding crowding and reducing agents. In addition to technological advances, we show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We also show that iSAT can be used for studying ribosome assembly. We anticipate that iSAT will aid studies of ribosome biogenesis and the construction of artificial cells.
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