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Open Access Technical Notes

A genetic replacement system for selection-based engineering of essential proteins

Sonja Billerbeck and Sven Panke*

Author Affiliations

ETH Zürich, Department for Biosystems Science and Engineering (D-BSSE), Mattenstrasse 26, 4058, Basel, Switzerland

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Microbial Cell Factories 2012, 11:110  doi:10.1186/1475-2859-11-110

Published: 16 August 2012

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.