A genetic replacement system for selection-based engineering of essential proteins
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Date
2012
Publication Type
Journal Article
ETH Bibliography
yes
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Abstract
Background
Essential 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.
Results
We 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.
Conclusions
We 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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published
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Book title
Journal / series
Volume
11
Pages / Article No.
110
Publisher
BioMed Central
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Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
Essential gene; Kanamycin resistance cassette; Chaperonin GroEL; Complementation vector; araBAD Promoter
Organisational unit
03602 - Panke, Sven / Panke, Sven