Department of Biology, Indiana University, Bloomington, Indiana, USA.
Department of Biology, Indiana University, Bloomington, Indiana, USA
Appl Environ Microbiol. 2020 May 5;86(10). doi: 10.1128/AEM.02893-19.
Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved -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 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 system will help enhance the flexibility of future transposon modification and application in and other organisms. 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 delivery vehicle in Moreover, the Tn 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.
随机转座子诱变是一种强大且无偏的遗传方法，可用于回答基础生物学问题。在这里，我们介绍了一种改进的基于转座子的系统，该系统在繁殖过程中具有更高的稳定性，并具有广泛的诱变应用。我们使用低拷贝数质粒作为转座子传递载体，这在载体构建和繁殖过程中降低了意外重组的频率。我们生成了各种转座子，允许基因缺失或人工过表达，每种转座子都与四种不同抗生素抗性标记之一结合。此外，我们还提供了由于转录或翻译偶联而报告基因/蛋白质表达的转座子。我们相信，Tn 系统将有助于提高未来转座子修饰和在和其他生物体中的应用的灵活性。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，我们提高了在中的 载体的稳定性。此外，Tn 转座子系统将通过增加抗生素抗性标记的数量以及生成报告蛋白质翻译和亚细胞定位的无偏 GFP 融合的能力，改善正向遗传方法的应用。
