@article{mbs:/content/journal/micro/10.1099/mic.0.051623-0, author = "Gestal, Alicia M. and Liew, Elissa F. and Coleman, Nicholas V.", title = "Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology", journal= "Microbiology", year = "2011", volume = "157", number = "12", pages = "3349-3360", doi = "https://doi.org/10.1099/mic.0.051623-0", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.051623-0", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", abstract = "Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (GmR or Lac+) were obtained at frequencies ranging from 101 to 106 c.f.u. (µg DNA)−1. Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter PC were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.", }