PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized non-ribosomal proteins

Abstract Building a universal biochemical constructor, an autonomously self-replicating bio-chemical system, is a major challenge in synthetic biology. The PURE cell-free system is an ideal starting point for exploring self-regeneration, and its 36 non-ribosomal proteins constitute the primary macromolecular components that must be regenerated. Here, we demonstrate that the PURE system can be reconstituted from proteins synthesized by PURE itself. We first show that each of the 36 non-ribosomal proteins can be individually synthesized in PURE. We then purify the PURE synthesized proteins as pooled subsets and reconstitute a fully functional PURE system by combining the subsets. Finally, we show that all 36 non-ribosomal PURE proteins can be synthesized simultaneously in a single PURE reaction and, after purification, can reconstitute a functional PURE system. Together, these results establish that the non-ribosomal protein components of the PURE system can be self-regenerated, representing a critical step toward the realization of a universal biochemical constructor.