Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology
Open Access
- 31 January 2013
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 41 (5), 3471-3481
- https://doi.org/10.1093/nar/gkt052
Abstract
A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.Keywords
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