偶联介导的多功能位点特异性单拷贝萤光素酶融合系统的开发。

IF 0.8 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal of General and Applied Microbiology Pub Date : 2024-05-02 Epub Date: 2023-11-07 DOI:10.2323/jgam.2023.10.001
Akinori Kato
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引用次数: 0

摘要

有许多报告系统可用于细菌中的基因表达分析。然而,至少在沙门氏菌中,目前还没有一种可以精确整合在染色体上感兴趣的启动子或基因后面的通用且简单的萤光素酶报告系统。来自发光光弹菌的萤光素酶操纵子luxCDABE具有几个优点,包括亮度、宽线性范围、在大多数细菌中不存在、在高温下的稳定性以及测定不需要添加底物。在此,开发了一种偶联介导的位点特异性单拷贝萤光素酶融合系统。将含有条件复制来源R6Kgγ、FRT-luxCDABE和KmR标记的报告质粒设计为掺入表达FLP的细胞染色体上感兴趣的启动子或基因后面的FRT位点。然而,当将该报告质粒直接电穿孔到这样的肠炎沙门氏菌菌株中时,没有出现菌落,这可能是由于这种相对较大的质粒DNA的转化效率较低。同时,成功地将同一报告质粒作为含有FRT的偶联转移质粒的插入物从交配的大肠杆菌菌株引入并启动到同一受体肠炎沙门氏菌菌株以及科塞里柠檬酸杆菌。证实了来自构建的wzc-luxCDABE菌株的RcsB依赖性诱导发光。该系统对于检测非常低水平的转录是可行的,即使在相对难以遗传操作的革兰氏阴性细菌物种中也是如此。
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Development of conjugation-mediated versatile site-specific single-copy luciferase fusion system.

There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and KmR marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.

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来源期刊
Journal of General and Applied Microbiology
Journal of General and Applied Microbiology 生物-生物工程与应用微生物
CiteScore
2.40
自引率
0.00%
发文量
42
审稿时长
6-12 weeks
期刊介绍: JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.
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