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Nucleic Acids Res. 2013 Mar 1;41(5):3471-81. doi: 10.1093/nar/gkt052. Epub 2013 Jan 31.

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology.

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1
Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

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.

PMID:
23371936
PMCID:
PMC3597704
DOI:
10.1093/nar/gkt052
[Indexed for MEDLINE]
Free PMC Article
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