Expression optimization and synthetic gene networks in cell-free systems

David K. Karig, Sukanya Iyer, Michael L. Simpson, Mitchel J. Doktycz

Research output: Contribution to journalArticlepeer-review

98 Scopus citations

Abstract

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

Original languageEnglish
Pages (from-to)3763-3774
Number of pages12
JournalNucleic Acids Research
Volume40
Issue number8
DOIs
StatePublished - Apr 2012

Funding

The authors thank Dr Dale A. Pelletier and Dr Jennifer Morrell-Falvey for helpful comments. This research was performed at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. The Center for Nanophase Materials Sciences that is sponsored by the Scientific User Facilities Division, Office of Science, U.S. Department of Energy (to D.K.K., M.L.S., M.J.D.) and National Institutes of Health (EB000657 to S.I. and M.J.D.); Funding for open access charge: National Institutes of Health (grant number EB000657).

FundersFunder number
Scientific User Facilities Division
National Institutes of Health
U.S. Department of EnergyDE-AC05-00OR22725
National Institute of Biomedical Imaging and BioengineeringR01EB000657
Office of Science
Oak Ridge National Laboratory

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