Expression Optimization and Synthetic Gene Networks in Cell-free Systems

David K. Karig,1 Sukanya Iyer,2,3 Michael L. Simpson,1,4,5 Mitchel J. Doktycz,1,2

1-Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Bethel Valley Road, Oak Ridge, Tennessee 37831, USA
2-Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA
3-Graduate Program in Genome Science and Technology 37996, University of Tennessee, Knoxville, Tennessee, USA
4-Department of Materials Science and Engineering, University of Tennessee, Knoxville, Tennessee, USA 37996-2010, USA
5-Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37996-2010, USA


We demonstrated the construction of cell-free genetic regulatory elements, and then used these elements to explore different approaches for composing regulatory systems . Ultimately, this led to the implementation of inducible negative feedback architectures in a cell-free context. Given the prevalence of negative feedback motifs in natural gene networks, our cell-free negative feedback constructs represent an invaluable building block for the development of more complex synthetic nanoscale systems such as nanofabricated “cell-mimics.”


The Grand Challenge for Nanoscience is to master energy and information on the nanoscale to create new technologies with capabilities rivaling those of living systems . Conversely, synthetic biology pursues a similar goal, but uses living cells. A synergistic approach is to use cell-like strategies in synthetic cell-free structures as has been presented in previous Center for Nanophase Materials Sciences (CNMS) research. Our demonstration of inducible negative feedback and the approaches used for optimization are essential steps in building more complex molecular information processing elements in synthetic nanoscale systems. Furthermore, by showing that the feedback systems developed in a cell-free context could be implemented in live E. coli, we illustrated the potential for interfacing systems developed in vitro to living organisms for application purposes. The presented synthetic biology efforts embody an important step towards harnessing biological strategies for designing complex function in a bottom-up fashion in synthetic nanoscale systems.


DKK, MLS, and MJD acknowledge support from the CNMS that is sponsored by the Scientific User Facilities Division, Office of Science, U.S. Department of Energy. SI and MJD also acknowledge support from the National Institutes of Health, grant number EB000657. 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.

iKarig D.K., Iyer S., Simpson M.L., Doktycz M.J., “Expression Optimization and Synthetic Gene Networks in Cell-free Systems,” Nucleic Acids Research. Published online Dec. 17, 2011 doi: 10.1093/nar/gkr1191

iiMatter and Energy: Five Challenges for Science and the Imagination, A Report from the Basic Energy Sciences Advisory Committee, Chapter 5, REALIZING THE DREAM OF NANOSCIENCE: ENERGY AND INFORMATION ON THE NANOSCALE