Random Mutagenesis

A library of B. thailandensis transconjugants was generated as follows. Plasmid pIT2 carrying the ISlacZ/hah transposon was transferred in B. thailandensis E264 by conjugation with E. coli χ7213 (asd-) strain (Kang et al., 2002; Jacobs et al., 2003). NB agar supplemented with 4% glycerol and tetracycline was used for selection of transposants. The screening for rhamnolipid production was achieved using atomized mineral oil spraying (Burch et al., 2010), with a few modifications. Sudan Red dye (0.5%) was added to mineral oil to provide a better contrast. The presence of a halo surrounding colonies indicates the production of rhamnolipids caused by the amphiphilic properties of surfactants; the diameter of halos around colonies was measured and compared to a WT control. Clones with larger halos were selected as potential candidates for enhanced rhamnolipid production.

Total DNA was extracted from bacterial cultures using a mechanical lysis method, as previously described (Durand et al., 2015). DNA concentrations were estimated using the Quant-iT^TM PicoGreen^® dsDNA Assay Kit (Invitrogen, Life Technologies, Burlington, ON, Canada) following the instructions of the manufacturer. Total genomic DNA from the selected mutants were pooled together and sent to the McGill University and Génome Québec Innovation Centre for transposon insertion sequencing (MiSeq Illumina). Generated Tn-Seq reads were analyzed as follows: sequences were trimmed in order to remove the 3′ bases from the adaptor used for sequencing; only the 4 last bases from the cassette were conserved: TCAG. All the resulting sequences were alphabetically sorted and clustered. For each cluster, a unique consensus sequence was determined and sequence alignments with B. thailandensis E264 genome were performed on www.burkholderia.com, allowing the identification of the insertion site.