sentence: This ligation mixture was then directly transformed into the wild ‐ type strain 168CA and colonies containing the correct integration of the entire spec ‐ P ‐ lacI construct were verified using PCR.The spoIIIE36 mutation [ which we here also denote as spoIIIE ( frozen ) ] containing the G1285 => A transition in the spoIIIE ORF , originally isolated by Piggot ( 1973 ; Wu and Errington , 1994 ) was introduced into the chromosome of B. subtilis wild ‐ type strain 168CA by fusing PCR products of the spoIIIE gene generated from chromosomal DNA of the spoIIIE36 mutant strain 36.3 with primer pairs oTK53 / oTK54 , oTK55 / oTK56 and oTK59 / oTK61 , and a PCR product containing the kanamycin resistance gene was PCR amplified using oTK57 / oTK58 from plasmid pHT21 ( Trieu ‐ Cuot and Courvalin , 1983 ) , in a single reaction by means of overlap ‐ extension PCR .