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Author Sven, Panke ♦ Sonja, Billerbeck
Source Directory of Open Access Journals (DOAJ)
Content type Text
Publisher BioMed Central
File Format HTM / HTML
Date Created 2013-03-12
Copyright Year ©2012
Language English
Subject Domain (in LCC) QR1-502
Subject Keyword Science ♦ Microbiology
Abstract 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 10 <sup>7</sup> transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 10 <sup>5</sup>-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.
ISSN 14752859
Age Range 18 to 22 years ♦ above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study
Learning Resource Type Article
Publisher Date 2012-08-01
e-ISSN 14752859
Journal Microbial Cell Factories
Volume Number 11
Issue Number 1
Starting Page 110


Source: Directory of Open Access Journals (DOAJ)