Figure 1

Overview on the transformation-based replacement of essential genes. A: Plasmids required for establishment of the selection system B: General procedure: The essential target gene adk is replaced by an antibiotic resistance cassette while pKOCOMP-adk complements for the chromosomal loss. Plasmid pKOCOMP can be conditionally eliminated by co-expression of I-Sce I nuclease from helper plasmid pI-Sce I. During elimination, cells are made electrocompetent and are transformed with a variant or library under investigation to evaluate functionality or to select for functional library members which can complement for the loss of pKOCOMP.