posted on 2022-01-17, 06:03authored byAngelo
Cardoso Batista, Antoine Levrier, Paul Soudier, Peter L. Voyvodic, Tatjana Achmedov, Tristan Reif-Trauttmansdorff, Angelique DeVisch, Martin Cohen-Gonsaud, Jean-Loup Faulon, Chase L. Beisel, Jerome Bonnet, Manish Kushwaha
The
use of linear DNA templates in cell-free systems promises to
accelerate the prototyping and engineering of synthetic gene circuits.
A key challenge is that linear templates are rapidly degraded by exonucleases
present in cell extracts. Current approaches tackle the problem by
adding exonuclease inhibitors and DNA-binding proteins to protect
the linear DNA, requiring additional time- and resource-intensive
steps. Here, we delete the recBCD exonuclease gene
cluster from the Escherichia coli BL21
genome. We show that the resulting cell-free systems, with buffers
optimized specifically for linear DNA, enable near-plasmid levels
of expression from σ70 promoters in linear DNA templates without
employing additional protection strategies. When using linear or plasmid
DNA templates at the buffer calibration step, the optimal potassium
glutamate concentrations obtained when using linear DNA were consistently
lower than those obtained when using plasmid DNA for the same extract.
We demonstrate the robustness of the exonuclease deficient extracts
across seven different batches and a wide range of experimental conditions
across two different laboratories. Finally, we illustrate the use
of the ΔrecBCD extracts for two applications:
toehold switch characterization and enzyme screening. Our work provides
a simple, efficient, and cost-effective solution for using linear
DNA templates in cell-free systems and highlights the importance of
specifically tailoring buffer composition for the final experimental
setup. Our data also suggest that similar exonuclease deletion strategies
can be applied to other species suitable for cell-free synthetic biology.