Cell-free expression of RuBisCO for ATP production in the synthetic cells.

Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested.

Progresses in Cell-Free In Vitro Evolution.

Biopolymers, such as proteins and RNA, are integral components of living organisms and have evolved through a process of repeated mutation and selection. The technique of "cell-free in vitro evolution" is a powerful experimental approach for developing biopolymers with desired functions and structural properties. Since Spiegelman's pioneering work over 50 years ago, biopolymers with a wide range of functions have been developed using in vitro evolution in cell-free systems.

In Vitro Transcription/Translation-Coupled DNA Replication through Partial Regeneration of 20 Aminoacyl-tRNA Synthetases.

The in vitro reconstruction of life-like self-reproducing systems is a major challenge in in vitro synthetic biology. Self-reproduction requires regeneration of all molecules involved in DNA replication, transcription, and translation. This study demonstrated the continuous DNA replication and partial regeneration of major translation factors, 20 aminoacyl-tRNA synthetases (aaRS), in a reconstituted transcription/translation system (PURE system) for the first time.