A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level
A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level
复制标题
DOI：
10.2144/000114317
复制
发表时间：
2015-08-01
期刊：
影响因子：
2.7
通讯作者：
Heermann, Ralf
中科院分区：
文献类型：
--
作者：
Glaeser, Angela;Heermann, Ralf
在单细胞水平上测定报告基因活性是分析细菌中异质基因表达的先决条件。昆虫致病性肠细菌Photorhabdus luminescens是一种出色的生物体，可以研究异质性，因为它以两种表型不同的形式存在，称为主要和次要变体。这些细菌缺乏一种用于生成报告基因基因稳定基因组整合的工具，这阻碍了单细胞水平上启动子活动的可靠数据集的获取。因此，我们生成了一个名为PPINT-MCHERRY的质粒工具，用于轻松稳定地引入MCHERRY记者基因上游的基因片段，然后将质粒稳定地整合到RPME/GLMS基因组中的P. luminescens基因组中。我们证明，在P. luminescens中，必须进行单细胞分析的报告基因的基因组整合，因为质粒传播的报告基因基因模仿了异质性，因此与这些细菌不适用于这些细菌，而与它们在其他细菌中的使用相比像大肠杆菌一样。
Determination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically different forms, called the primary and secondary variant. A tool for generating stable genomic integrations of reporter genes has been lacking for these bacteria, and this has hampered the acquisition of reliable data sets for promoter activities at the single-cell level. We therefore generated a plasmid tool named pPINT-mCherry for the easy and stable introduction of gene fragments upstream of an mCherry reporter gene followed by stable integration of the plasmid into the P. luminescens genome at the rpmE/glmS intergenic region. We demonstrate that the genomic integration of reporter genes for single-cell analysis is necessary in P. luminescens since plasmid-borne reporter genes mimic heterogeneity and are therefore not applicable in these bacteria, in contrast to their use in single-cell analysis in other bacteria like Escherichia coli.