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. 2015 Feb 4;15(1):17. doi: 10.1186/s12866-015-0354-3

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© Bruckbauer et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Promoter identification and capture using mini-Tn 5/7-lux elements. Step 1 involves random Tn5 transposition into the chromosome of the bacterial host of interest (target bacterium) after conjugal transfer of the mini-Tn5/7-lux delivery plasmid. This plasmid also contains the Tn5 transposase encoding tnpA gene. TnpA acts on the Tn5 mosaic ends (ME), which results in random mini-Tn5/7-lux transposition into the bacterial chromosome, including insertion in a gene where the Photorhabdus luminescens luxCDABE operon is transcribed from the gene’s promoter (P). The mini-Tn7 element, i.e. sequences flanked by the Tn7 left (Tn7L) and right (Tn7R) ends, located on the mini-Tn5/7-lux delivery plasmid does not transpose in this step because the delivery plasmid does not encode Tn7 transposase. Mini-Tn5/7-lux chromosomal insertion is stable because the chromosomally-integrated elements do neither encode Tn5 nor Tn7 transposase. In step 2, chromosomal DNA of in this example kanamycin resistance (Km^r) and light-emitting transformants is isolated and digested with a restriction enzyme (R) that does not cleave within the transposed element. In step 3, chromosomal DNA fragments are religated and a plasmid containing the R6K origin of replication (ori _R6K) and origin of conjugal transfer (oriT) is recovered by transformation of a pir ⁺ E. coli host and selecting Km^r transformants. Sequencing of plasmid DNA with a luxC-specific primer (P2385) will reveal the transposon insertion site and putative promoter sequences. The Km^r selection marker contained on the chromosomally integrated mini-Tn7-lux elements is flanked by Flp recombinase target (FRT) sites for optional marker excision with Saccharomyces cerevisiae Flp recombinase.