Tn5tac1 insertion polarity in Escherichia coli

The Tn5-derived transposon Tn5tac1 carries the strong tac promoter (Ptac) facing outward at one of its ends. Expression of Ptac is under the control of the lacIq gene, also contained within the transposon. By inserting Tn5tac1 upstream from a promoterless galK gene we determined the basal level of transcription from both ends of the transposon in the absence of IPTG to be about 4% relative to the lactose promoter (Plac). As a result, derivatives of strain N100 containing these plasmids produce red colonies in MacConkey-galactose plates. Deletion of the BamHI fragment including Ptac causes galactokinase levels to drop to less than 1% of Plac, enough to render white colonies in MacConkey-galactose plates. Thus, Tn5tac1 can be used for genetic analysis under conditions in which it shows no polarity (+ IPTG), low polarity (- IPTG), or strong polarity (delta Ptac).