ArticleMicrobial cell factories2012
A genetic replacement system for selection-based engineering of essential proteins.
Article in Microbial cell factories, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 6 citations in OpenAlex.
- Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides.Applied and environmental microbiology · 2018Article
- Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.BMC biology · 2017Article
- Establishment of a markerless mutation delivery system in Bacillus subtilis stimulated by a double-strand break in the chromosome.PloS one · 2013Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundEssential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches.
resultsWe established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation.The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains.
conclusionsWe propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies.
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