Supplementary Data for: PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized proteins

Changes in this version (v3): Updated Figure numbers. Abstract: Building a universal biochemical constructor, an autonomously self-replicating biochemical system, remains a challenge in synthetic biology. The PURE cell-free system is a suitable starting point for exploring self-regeneration, and its 36 non-ribosome 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-ribosome 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-ribosome 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-ribosome proteins components of the PURE system can be self-regenerated, representing an essential step toward the realization of a universal biochemical constructor.