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ACS Synth Biol ; 10(7): 1615-1624, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34161082

RESUMO

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the in vitro operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In Escherichia coli extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to Vibrio natriegens extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.


Assuntos
Ácidos Nucleicos Livres/genética , Escherichia coli/genética , Biologia Sintética/métodos , Vibrio/genética , Ácidos Nucleicos Livres/química , Conformação de Ácido Nucleico , Estudo de Prova de Conceito
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