Schematic overview of all insertion sites of the regulatable E. coli candidates found in the screens. “IE” depicts the insertion site of the element. The black arrow represents the direction of the tetA promoter.

M9 minimal medium agar plates containing 2% glucose as a carbon source with and without 0.4 μM atc (+atc and −atc, respectively) showing the growth phenotypes of the regulatable E. coli candidates. Letters A to M refer to the candidates described in Table 3.

Test of the regulatory function of the insertion element InsTet^G−1 cloned in front of a promoterless lacZ gene and cotransformed with TetR-expressing plasmids. The large regulatory window of InsTet^G−1 was determined by measurements of β-gal activity. The percent β-gal activity can be seen on the y axis. The left panel shows the results for E. coli and the right panel shows results for Salmonella serovar Typhimurium with atc (black columns) and without atc (white columns). Measurements without a regulator (−) represent the 100% values, with 1,120 Miller units (MU) (with atc) and 1,200 MU (without atc) for E. coli and 2,350 MU (with atc) and 1,720 MU (without atc) for Salmonella serovar Typhimurium. TetR and revTetR^r2 represent the two TetR variants expressed from pWH1411.

Architecture of the insertion element InsTet^G−1. The entire element is shown schematically. It is 1,376 bp long and flanked by 19-bp recognition sites for hyperactive Tn5 transposase (ME). The modified tet P_A promoter (right-directed gray arrow) on the one side containing two tet operators (gray boxes) can drive transcription of downstream genes. The kanamycin resistance gene (aphAIII) allows the selection of candidates. A bidirectional transcription terminator from Tn10 (stem-loop) upstream of the Km^r cassette blocks ingoing and outgoing transcription, and stop codons in all three forward reading frames (“S”) terminate possible translation from upstream. Restriction sites for NcoI at both ends of the kanamycin cassette facilitate the exchange of the selection marker, and restriction sites for PvuII at both ends of the insertion element enable blunt cutting for in vitro transposome formation. The sequence of the modified tetracycline-dependent control region of transposon Tn10 is displayed underneath. One base pair mutation (black boxed letters) shuts down the activity of both P_R promoters without influencing the P_A promoter. O₁/O₂ and the underlined sequences indicate the operator sequences, and the −35 and −10 regions of the P_A promoter are indicated by black boxes. The arrow depicts the transcription start site of the promoter.