2025 AIChE Annual Meeting

(610d) Systems-Level Engineering of a Reconstituted One-Pot Pure System for Robust, Reproducible, and High-Yield Cell-Free Gene Expression

Authors

Yan Zhang - Presenter, Georgia Institute of Technology
Matas Deveikis, Imperial College London
Yanping Qiu, California Institute of Technology
Lovisa Björn, California Institute of Technology
Tsui-Fen Chou, California Institute of Technology
Paul Freemont, Imperial College London
Cell-free expression systems, which enable gene transcription and translation outside living cells, have become foundational tools in synthetic biology. Reconstituted systems like the One-Pot PURE platform offer greater control over the gene expression environment than lysate-based alternatives, allowing precise tuning of both the expression machinery and energy regeneration pathways. However, since the One-Pot PURE approach was introduced in 2019, achieving consistent and high-yield performance has remained a key challenge.

In this work, we present a systems-level engineering strategy to improve the robustness, reproducibility, and productivity of the One-Pot PURE system. We systematically identified and mitigated key sources of variability in system performance. During recombinant expression of the PURE proteins, we found that genetic instability led to spontaneous protein dropouts, which we addressed by optimizing growth media using catabolite repression. Additionally, we discovered that several essential proteins were poorly expressed in E. coli M15—the host strain originally recommended for PURE protein production. Replacing this strain with a more suitable expression host significantly increased protein abundance and improved overall system output. Beyond protein composition, we found that differences in energy formulations—likely modulated by tRNA availability—could partially compensate for deficiencies in PURE protein content. These insights suggest that engineering the tRNA pool may offer an additional layer of control to boost protein synthesis efficiency.

Altogether, this work highlights the complex biochemical interplay underlying reconstituted cell-free systems and provides a reproducible, high-yield framework for PURE-based gene expression. Our findings advance the development of robust, minimal gene expression platforms for a wide range of synthetic biology applications.