The Escherichia coli ribosome is a 2.4 MDa molecular machine made up of 57 RNA and protein parts. To support bacterial log phase growth with a 40 minute doubling time, ribosomes must be produced at a rate of ~8 ribosomes per second. Once assembled, these ribosomes can synthesize sequence-defined polymers of amino acids with elongation rates of ~15 amino acids per second and an accuracy of ~99.99%. The rapid rate of synthesis and incredible catalytic capability motivate efforts to understand the systems biology of ribosome biogenesis and function, as well as to create engineered variants with altered function. Indeed, the construction of engineered ribosomes has emerged as a defining challenge in synthetic biology, with transformative opportunities to build minimal cells, enable dual translation systems in cells, and catalyze a new paradigm for the synthesis and evolution of abiological polymers. Unfortunately, the requirement of cell viability severely limits the mutations that can be made to the ribosome.

We seek to address this critical limitation both in vivo and in vitro. In vivo, we have developed the first fully orthogonal ribosome-mRNA system by creating ribosomes with tethered subunits (termed Ribo-T). In vitro, we established a cell-free ribosome construction platform, termed iSAT (integrated synthesis, assembly, and translation of ribosomes) to build modified ribosomes in test tubes.