Sealable femtoliter chamber arrays for cell-free biology
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. Lastly, 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 to characterize CFPS gene circuits and their interactions with the cell-free environment.
- Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Publication Date:
- OSTI Identifier:
- Grant/Contract Number:
- Accepted Manuscript
- Journal Name:
- Journal of Visualized Experiments
- Additional Journal Information:
- Journal Issue: 97; Journal ID: ISSN 1940-087X
- MyJoVE Corp.
- Research Org:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org:
- USDOE Office of Science (SC)
- Country of Publication:
- United States
- 59 BASIC BIOLOGICAL SCIENCES cell-free; synthetic biology; microfluidics; noise biology; soft lithography; femtoliter volumes; bioengineering; Issue 97
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