TnFLX: a third-generation mariner-based transposon system for Bacillus subtilis

Random transposon mutagenesis is a powerful genetic tool to answer fundamental biological questions in an unbiased approach. Here, we introduce an improved mariner-based transposon system with higher stability, and with versatile applications. We take advantage of the lower frequency of unintended recombination during vector construction and propagation in a low copy number system in E. coli to improve construct integrity. 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 TnFLX system will help enhance flexibility of future transposon modification and application in Bacillus and other organisms.\n\nImportanceThe optimization of transposase encoding vectors in terms of stability during cloning and propagation is crucial for the reliable application of this system in any host organism. With an increased number of antibiotic resistance markers and the possibility to detect translational activity, the TnFLX transposon system will significantly help the implication of forward genetic methods in the field of cellular biology.