Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts.
ACS Synth Biol
; 11(2): 732-746, 2022 02 18.
Article
in En
| MEDLINE
| ID: mdl-35034449
ABSTRACT
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Escherichia coli
/
Exonucleases
Language:
En
Journal:
ACS Synth Biol
Year:
2022
Document type:
Article
Affiliation country:
France