In:
PLOS ONE, Public Library of Science (PLoS), Vol. 17, No. 4 ( 2022-4-7), p. e0266272-
Kurzfassung:
Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli ( E . coli ). Endogenous nucleases in E . coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E . coli , with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E . coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E . coli strain, enabling the creation of TXTL systems with other custom modifications.
Materialart:
Online-Ressource
ISSN:
1932-6203
DOI:
10.1371/journal.pone.0266272
DOI:
10.1371/journal.pone.0266272.g001
DOI:
10.1371/journal.pone.0266272.g002
DOI:
10.1371/journal.pone.0266272.g003
DOI:
10.1371/journal.pone.0266272.g004
DOI:
10.1371/journal.pone.0266272.g005
DOI:
10.1371/journal.pone.0266272.s001
DOI:
10.1371/journal.pone.0266272.s002
DOI:
10.1371/journal.pone.0266272.s003
DOI:
10.1371/journal.pone.0266272.s004
DOI:
10.1371/journal.pone.0266272.s005
DOI:
10.1371/journal.pone.0266272.s006
DOI:
10.1371/journal.pone.0266272.s007
DOI:
10.1371/journal.pone.0266272.s008
Sprache:
Englisch
Verlag:
Public Library of Science (PLoS)
Publikationsdatum:
2022
ZDB Id:
2267670-3
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