Sealable Femtoliter Chamber Arrays for Cell-free Biology |
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Authors: | Sarah Elizabeth Norred Patrick M Caveney Scott T Retterer Jonathan B Boreyko Jason D Fowlkes Charles Patrick Collier Michael L Simpson |
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Institution: | 1Bredesen Center, University of Tennessee, Knoxville;2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory;3Department of Materials Science and Engineering, University of Tennessee, Knoxville |
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Abstract: | Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment. |
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Keywords: | Bioengineering Issue 97 Cell-free synthetic biology microfluidics noise biology soft lithography femtoliter volumes |
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