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

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

SUBMITTER: Billerbeck S 

PROVIDER: S-EPMC3503863 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature