Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology.
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.
Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
James
J
Chappell
Kirsten
K
Jensen
Paul S
PS
Freemont
D011088:DNA Ligases / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004269:DNA, Bacterial; D004270:DNA, Circular; D011088:DNA Ligases