The kanamycin-selected cassette pDB284 could be exchanged for the zeocin-selected pDB299, that has no fmt, thus producing a full deletion mutant. (a) mDB123 was electroporated by pDB299 and plated on zeocin. Blue colonies were picked and analyzed by PCR using primers intEnd, OriEstart. The primers bind to pDB284 (and thus, the “parent strain” mDB123), producing a 1532 bp product, and to pDB299 (and to strains where a correct exchange occurred), producing a 830 bp product. All 4 blue colonies were correct transformants, and one of them (*) was chosen for continuation, and named mDB150 (Δfmt:hyg; attb:zeo:lacZ). (b) Four different PCR reaction [using primers strt/R1 (950 bp), mid1/mid2 (480 bp), mid1/R1 (750 bp), mid2/strt (670 bp)], all amplifying Mtbfmt, were performed on wt and mDB150. All reactions failed to detect presence of fmt in mDB150.