Fig. 2

mTn5 transposon mutagenesis using an IPTG-controlled conditional suicide plasmid. a Diagram of plasmid pSNC-mTn5. pSNC-mTn5 is a derivative of pMMB-repF that contains a Kan^R-tagged mTn5, a lac promoter-controlled hyperactive transposase gene (tnp^H), and a sacB counter selection marker (with its own promoter). OE and IE are outside and inside ends of the mTn5. b Plasmid and transposon retention frequencies in E. coli DH10B. A “+” symbol for IPTG indicates that the inducer was added to the liquid culture, and a “+” symbol for Suc, Cam, and Kan indicates that the chemicals were added to the plates. Black columns represent plasmid retention frequencies, and the blue column represents Tn5 retention frequency. Results were average of three independent experiments, and bars represent mean ± SD (*p < 0.0001 and **p = 0.0054 by unpaired t-test). (see Methods for details) (c) Colony restreaking. 150/150 Suc^RKan^R colonies of DH10B were found to be Kan^RCam^S and 50 are shown here. d Colony PCR of 10 restreaked clones in (c). Primer sets P1&P2 and P3&P4 detect Kan^R and repF, respectively. All were mTn5-positive and plasmid-negative. Primers P3 and P4 are a functional pair for PCR-amplification of the plasmid sequence (data not shown). e Plasmid and transposon retention frequencies in A. baylyi. Results were average of three independent experiments, and bars represent mean ± SD (*p = 0.024 and **p < 0.0001 by unpaired t-test). f Plasmid and transposon retention frequencies in P. aeruginosa. Results were average of three independent experiments, and bars represent mean ± SD (*p = 0.0013 and **p = 0.0038 by unpaired t-test). Colony restreaking and PCR analysis are shown in Additional file 2: Figure S2