Detection of recombinase activity in <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> cells were transformed with plasmids pFT74 (<b>A</b>) or pFT74^R-<i>attP</i> carrying a <i>skin</i>^<i>Cd</i>-less <i>sigK</i> gene and <i>attP</i> in both possible orientations (<b>C</b> and <b>E</b>). The resulting strains were then transformed with plasmids containing the <i>CD1231-Strep</i>, <i>CD1234-His</i>_<i>6</i> or both genes (“+” signs) under the control of an IPTG inducible promoter. <b>A, C and E</b>: schematic representation of the recombination event (<i>skin</i>^<i>Cd</i> excision in A, <i>skin</i>^<i>Cd</i> integration in C and DNA inversion in E), the expected recombination products and the size of the intact and recombined inserts between the BamHI and NotI sites. <i>attP</i>´denotes an inverted <i>attP</i> site. <b>B, D and F:</b> analysis of the recombinant events following pFT74 or pFT74^R-<i>attP</i> recovery from <i>E</i>. <i>coli</i> strains producing <i>CD1231-Strep</i>, <i>CD1234-His</i>_<i>6</i> or both genes (“+” signs) and digestion with BamHI and NotI. pFT74 and pFT74^R+<i>attP</i> from strains that did not produce <i>CD1231-Strep</i> or <i>CD1234-His</i>_<i>6</i> were used as controls for non-recombined and recombined <i>sigK</i>, respectively. The size of molecular marker (in bp) is indicated on the left side of the panels. The “+” sign at the bottom of the panels indicate clones where the expected recombination event has occurred (8 clones were analyzed).