Modular cell-free expression plasmids to accelerate biological design in cells

Karim, Ashty S.; Liew, Fungmin (Eric); Garg, Shivani; Vögeli, Bastian; Rasor, Blake J.; Gonnot, Aislinn; Pavan, Marilene; Juminaga, Alex; Simpson, Séan D.; Köpke, Michael; Jewett, Michael C.

doi:10.1093/synbio/ysaa019

Modular cell-free expression plasmids to accelerate biological design in cells

Journal Article · Wed Oct 14 00:00:00 EDT 2020 · Synthetic Biology

DOI:https://doi.org/10.1093/synbio/ysaa019· OSTI ID:1854008

^[1]; Liew, Fungmin (Eric) ^[2]; Garg, Shivani ^[2]; ^[3]; ^[3]; Gonnot, Aislinn ^[2]; Pavan, Marilene ^[2]; Juminaga, Alex ^[2]; Simpson, Séan D. ^[2]; Köpke, Michael ^[2]; Jewett, Michael C. ^[3]

Northwestern Univ., Evanston, IL (United States); Northwestern University
LanzaTech, Inc., Skokie, IL (United States)
Northwestern Univ., Evanston, IL (United States)

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

View Accepted Manuscript (DOE)

Research Organization:: Northwestern Univ., Evanston, IL (United States); Northwestern University, Evanston, IL (United States)

Sponsoring Organization:: Swiss National Science Foundation (SNSF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; SC0018249

OSTI ID:: 1854008

Alternate ID(s):: OSTI ID: 2298969

Journal Information:: Synthetic Biology, Journal Name: Synthetic Biology Journal Issue: 1 Vol. 5; ISSN 2397-7000

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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Modular cell-free expression plasmids to accelerate biological design in cells

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