A genetic replacement system for selection-based engineering of essential proteins.
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.
ETH Zürich, Department for Biosystems Science and Engineering-D-BSSE, Mattenstrasse 26, 4058 Basel, Switzerland.
Sonja
S
Billerbeck
Sven
S
Panke
D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020043:Genes, Essential; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D000263:Adenylate Kinase