ETOP 503689: Pre-optimized cell free lysates for rapid prototyping of genes and pathways

Technical Report · Mon Feb 12 04:00:00 EST 2024

DOI:https://doi.org/10.2172/2298929· OSTI ID:2298929

Alper, Hal ^[1]; Jewett, Michael ^[2]

---

1. Univ. of Texas, Austin, TX (United States)
2. Northwestern Univ., Evanston, IL (United States)

This ETOP project aimed to re-conceive how we engineer complex biological systems by linking pathway design, prospecting, and validation into an integrated framework. Specifically, our vision seeks to advance and interweave high-throughput cell-based systems and rapid cell-free technologies in a way suitable for automation and microdroplet manipulation in a DOE JGI user facility setting. The key technology to be investigated toward this vision is a cell-free platform for combinatorial assembly of pathways by mixing-and-matching crude cell lysates derived from a suite of flux-enhanced background strains, each enriched with pathway enzymes. Through this work, we have established rewired E. coli and S. cerevisiae cells suitable to generated rewired lysates. We have demonstrated that lysates derived from rewired cells can generate higher fluxes of products and can enable a more robust discovery method going from gene to function annotations. Finally, we demonstrate that these rewired lysates can be stored for up to a year and still retain function. Collectively, these technologies enable a more rapid screening approach for enzyme and pathway variants.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• DOE Contract Number: AC02-05CH11231
• OSTI ID: 2298929
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES