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Fig. 1 | Mobile DNA

Fig. 1

From: Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

Fig. 1

Characterization of the papillation assay using a strong inducible promoter. a. The Hsmar1 transposon (RC5096), which encodes a lacZ gene lacking transcription and translation signals and a kanamycin resistance marker (kanR), has been integrated in a non-transcribed region of a lac- E. coli strain. In absence of a vector encoding the transposase, the lacZ gene cannot be transposed in frame into an active open reading frame. The strain remains lac- and produces white colonies on plates containing X-gal. In presence of the transposase, the transposon can integrate in frame into the ORF of a transcribed gene, producing a lacZ fusion protein. The cell’s descendants will express lacZ and will appear as blue papillae on plates containing X-gal. Black arrow, promoter; open brackets, transposon ends; empty rectangle, transposase gene. For the mating-out assay, a chloramphenicol resistant derivative of the conjugative plasmid pOX38 is introduced into the reporter strain. Transposition of the kanR-marked transposon into the plasmid is detected by selecting transconjugants after mating with a recipient strain on chloramphenicol and kanamycin. b. An expression vector encoding no transposase (pMAL-c2X), Hsmar1 (pRC1721) or MBP-Hsmar1 (pRC880) transposase (t’ase) was transformed into the papillation strain and plated on different lactose and IPTG concentrations. Representative colonies of the papillation plates are shown. On some pictures, smaller colonies surrounding the main colony are visible. These satellite colonies appear only after several days of incubation when the ampicillin present on the plate has been degraded. They can be ignored because they do not contain any transposase expression plasmid. Part of this figure was previously published in [19] under the terms of the Creative Commons CC BY license. c. Quantification of the number of papillae per colony from single colonies. Average ± standard deviation of six representative colonies from the same biological replicate

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