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

Changes in this version (v2): Added raw data for Supplementary Figures 2 and 3. Included the underlying gel image for Supplementary Figure 2b. Abstract: Building a universal biochemical constructor, an autonomously self-replicating biochemical 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-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 a critical step toward the realization of a universal biochemical constructor.本版本（v2）更新内容如下： 新增补充图2与补充图3的原始数据。 纳入补充图2b的原始凝胶电泳图像。 摘要： 构建通用生化构建器——一种可自主自我复制的生化系统，是合成生物学领域的重大挑战。无细胞PURE系统（PURE cell-free system）是探索自我再生的理想起点，其包含的36种非核糖体蛋白是实现系统再生所需的核心大分子组分。本研究证实，可利用PURE系统自身合成的蛋白重新重构完整的PURE系统。首先，我们证明36种非核糖体蛋白中的每一种均可在PURE系统中单独合成。随后，我们将PURE系统合成的蛋白按混合亚组进行纯化，并通过组合这些亚组重构得到功能完整的PURE系统。最后，我们证实，可在单次PURE反应中同时合成全部36种非核糖体PURE蛋白，经纯化后即可重构得到具备功能的PURE系统。综上，本研究结果证实PURE系统的非核糖体蛋白组分可实现自我再生，这为实现通用生化构建器迈出了关键一步。