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

Abstract

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. First, we replicated each DNA that encodes one of the 20 aaRSs through aaRS expression from the DNA by serial transfer experiments. Thereafter, we successively increased the number of aaRS genes and achieved simultaneous, continuous replication of DNA that encodes all 20 aaRSs, which comprised approximately half the number of protein factors in the PURE system, except for ribosomes, by employing dialyzed reaction and sequence optimization. This study provides a step-by-step methodology for continuous DNA replication with an increasing number of self-regenerative genes toward self-reproducing artificial systems.

Investigation of Compatibility between DNA Replication, Transcription, and Translation for in Vitro Central Dogma

“…Despite the inhibition, DNA replication still reached approximately 40fold, which is sufficient to maintain a constant concentration in serial dilution experiments performed in previous studies. 27,29,30 Furthermore, the increased transcription and translation are beneficial for the construction of a selfregenerating system, which is one of the next important targets in the field of in vitro synthetic biology 31,33 and requires the synthesis of large amounts of RNA and proteins. The results obtained in this study provide useful knowledge and methods to achieve a self-regenerating artificial system in the future.…”

“…The in vitro reconstitution of cellular functions has proven to be a useful approach for understanding living systems and developing new biotechnologies. − One of the central functions of the cell is the replication and expression of genetic information, known as the central dogma, which comprises three reactions: transcription, translation, and DNA replication. These reactions have been partially reconstituted in vitro , both independently and in combination. − However, their efficiency is still far from being comparable to that in living cells, especially for the combined reaction, which hinders the development of a self-sustaining artificial system. ,− …”

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution

“…For example, it has been achieved that the elements of protein synthesis are expressed by the PURE system. 7,38,39 Elements for cell division such as membrane proteins and spatiotemporal regulators for the cell division plane are also synthesized by the PURE system in liposomes. 12,40−42 However, the conventional PURE system used in these studies lacks metabolic pathways, which are the other essential systems for self-reproduction.…”

“…Recent numerous attempts have reconstituted cellular functions using the PURE system. For example, it has been achieved that the elements of protein synthesis are expressed by the PURE system. ,, Elements for cell division such as membrane proteins and spatiotemporal regulators for the cell division plane are also synthesized by the PURE system in liposomes. ,− However, the conventional PURE system used in these studies lacks metabolic pathways, which are the other essential systems for self-reproduction. Since glycolysis is the most upstream metabolic pathway, the coupling between glycolysis and protein synthesis serves as a fundamental supplier of elements for self-replication such as phospholipids, amino acids, and nucleic acids as well as the role of ATP and NADH regeneration.…”

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA

“…Recent advancements in the quest for the minimal synthetic cell have been substantial, especially in critical areas such as physicochemical homeostasis, − DNA replication, − membrane growth, , and membrane scission. ,− The emerging synthetic cell will ultimately harness its own gene expression machinery to regulate these complex features. In vitro transcription-translation (in vitro transcription and translation (IVTT)) is a well-established technology for the synthesis of proteins from plasmids and linear DNA encoding for a small number of genes. − Previous reports on phage genomes and large plasmids have shown that it is possible to express in the order of tens to a few hundred genes using DNA templates longer than 100 kbp. , Whereas these examples are excitingly close to a formerly postulated minimal genome (113 kbp with 151 genes), top-down experimental work on Mycoplasma established that the current minimal synthetic genome requires at least 493 genes. , From a bottom-up perspective, it is conceivable that the construction of synthetic cells requires similarly sized genomes, and these will need to be expressed in vitro.…”