Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, CH-1015 Lausanne, Switzerland.
Genes (Basel). 2019 May 22;10(5):394. doi: 10.3390/genes10050394.
Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen . The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.
在这里，我们描述了三个整合载体 pPEPX、pPEPY 和 pPEPZ 的构建，用于研究机会性病原体 。这些构建的载体被命名为肺炎链球菌工程平台（PEP）的 PEP，采用 IPTG 诱导启动子和 BglBrick 和 BglFusion 兼容的多克隆位点，允许快速且可互换的克隆。PEP 质粒在 中复制，并具有与大量肺炎链球菌株同源的整合位点，包括最近的临床分离株。此外，还提供了多种抗生素抗性标记物的选择，甚至允许在多药耐药的临床分离株中进行选择。我们测试了这些 PEP 载体的转化效率及其同时表达的能力。三个 PEP 载体中的两个在整合区域与超过一半的 基因组具有同源性。基于同源区域的长度，不同的 PEP 载体的转化效率存在差异，但它们都具有很高的可转化性，并且可以在 D39V 菌株中同时进行整合。用于肺炎链球菌克隆的载体是研究人员进行广泛应用的重要工具。描述的 PEP 载体尤其有用，因为它们的设计允许在载体之间轻松转移基因，并整合到染色体的转录沉默区域。此外，我们还成功地从 PEP 载体中生产了几种新的光谱明显不同的荧光蛋白（mTurquoise2、mNeonGreen 和 mScarlet-I）。PEP 载体和新描述的荧光蛋白将扩展肺炎球菌研究人员的遗传工具包，有助于未来的发现。
