Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology
- Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London SW7 2AZ, UK and Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
- ↵*To whom correspondence should be addressed. Tel: +44 207 5945327; Fax: +44 207 5943057; Email: p.freemont{at}imperial.ac.uk
- Received October 16, 2012.
- Revision received January 7, 2013.
- Accepted January 12, 2013.
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.
- © The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
