Fig. 4

Application of the validation methods in a strain engineered at a different chromosome locus. a The scheme depicts the replacement of the endogenous non-essential galK gene with an arabinose inducible primase gene (pBad-DnaG) along with a CmR gene through λ-Red recombination. The positions of the flanking primers for the galK gene (PgalKfw and PgalKrv) are marked, and the corresponding product lengths from PCR are indicated, along with the probed region in chromosome and the expected band size from Southern blot analysis. b The Southern blot results for the different experiments. Lane 1: the AB1157 sample in lane 1 has no insert, as expected. Lane 2: the pBad-DnaG strain obtained through recombineering has multiple bands (mainly at ~9 kbp, 7 kbp, 6.5 kbp, and 2.5 kbp). Lane 3: the pBad-DnaG strain after P1 phage transduction of the intended locus into the wild type AB1157 strain displays one band (~9 kbp) at the right fragment size, showing that the extraneous insertions can be removed in the final strain using this approach. c A flow diagram summarizing the sequence of the various validation techniques that should be performed prior to subsequent usage of the chromosomally engineered E.coli strain