Open AccessTn FLX : a Third-Generation mariner -Based Transposon System for Bacillus subtilis
Author(s) -
Felix Dempwolff,
Sandra Sánchez,
Daniel B. Kearns
Publication year - 2020
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.02893-19
Subject(s) - transposable element , transposase , transposon mutagenesis , biology , tn3 transposon , bacillus subtilis , sleeping beauty transposon system , genetics , plasmid , mutagenesis , escherichia coli , p element , computational biology , gene , mutation , genome , bacteria
Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner -based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the Tn FLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms. IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli Moreover, the Tn FLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.