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 an enhanced stability during propagation and versatile applications in mutagenesis. We used a low copy number plasmid as a transposon delivery vehicle that affords a lower frequency of unintended recombination during vector construction and propagation to improve the construct integrity in E. 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 TnFLX 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 increase the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased GFP fusions to report on protein translation and sub-cellular localization.

随机转座子诱变是回答基本生物学问题的有力且公正的遗传方法。在这里，我们介绍了一种改进的基于水手的转座子系统，该系统在繁殖过程中具有增强的稳定性，并在诱变中具有广泛的应用。我们使用低拷贝数质粒作为转座子传递载体，在载体构建和繁殖过程中提供了较低的意外重组频率，以改善大肠杆菌中的构建体完整性。我们产生了多种转座子，它们与四种不同的抗生素抗性标记之一结合在一起，可进行基因破坏或人工过表达。此外，我们提供的转座子将报告由于转录或翻译偶联而导致的基因/蛋白质表达。我们相信，Tn FLX系统将有助于增强将来的转座子修饰和在芽孢杆菌和其他生物中的应用灵活性。

重要性

转座酶编码载体在克隆和繁殖过程中的稳定性对于转座子的可靠应用至关重要。在这里，我们提高了水手运输载体在大肠杆菌中的稳定性。此外，Tn FLX转座子系统将通过增加数量的抗生素抗性标记物以及产生无偏的GFP融合蛋白以报告蛋白质翻译和亚细胞定位的能力，来改善正向遗传方法的应用。