双歧杆菌氯霉素乙酰转移酶报告系统的构建及其在启动子和终止子评价中的应用。

IF 3.1 4区 医学 Q2 Agricultural and Biological Sciences
Bioscience of Microbiota, Food and Health Pub Date : 2021-01-01 Epub Date: 2021-01-19 DOI:10.12938/bmfh.2020-070
Tomoya Kozakai, Yoko Shimofusa, Izumi Nomura, Tohru Suzuki
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引用次数: 3

摘要

报告基因分析系统是研究基因表达机制的重要工具。在双歧杆菌的情况下,已经开发了几个方便和敏感的报告系统。在这里,我们利用金黄色葡萄球菌的氯霉素乙酰转移酶基因(cat)开发了一个新的双歧杆菌报告系统。该酶在化学计量上产生游离辅酶a - sh,使用2-硝基苯甲酸(DTNB)进行Ellman试验定量分析。所制得的2-硝基-5-噻吩酸酯(TNB2-)呈淡黄色,最大吸光度在412 nm处。我们还构建了一个新的pBCMAT质粒系列,用于双歧杆菌的CAT检测,以评估启动子和终止子。对长双歧杆菌NCC2705启动子的分析表明,使用这些启动子的CAT测定是定量的,测量范围宽,稳定。此外,该试验对几种双歧杆菌种类也有用,包括长双歧杆菌、短双歧杆菌和青少年双歧杆菌。与厌氧条件下使用的荧光蛋白evoglow-Bs2相比,CAT实验显示其背景活性约为0.25%。在CAT分析中,我们鉴定出长芽孢杆菌NCC2705的11个启动子和12个终止子。编码核糖体蛋白、延伸因子和转移rna的基因具有强启动子,而包含强茎环和多u尾结构的终止子往往具有高活性。虽然上述启动子比终止子对表达活性的贡献更大,但被测终止子之间活性的最大倍数差异约为17倍。启动子中-10 box和5'-UTR的修饰以及终止子茎环周围结构的修饰都会影响表达水平。这些结果表明,CAT分析是有用的双歧杆菌基因表达的各种分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman's test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5'-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.

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来源期刊
Bioscience of Microbiota, Food and Health
Bioscience of Microbiota, Food and Health Immunology and Microbiology-Applied Microbiology and Biotechnology
CiteScore
5.50
自引率
3.20%
发文量
24
期刊介绍: Bioscience of Microbiota, Food and Health (BMFH) is a peer-reviewed scientific journal with a specific area of focus: intestinal microbiota of human and animals, lactic acid bacteria (LAB) and food immunology and food function. BMFH contains Full papers, Notes, Reviews and Letters to the editor in all areas dealing with intestinal microbiota, LAB and food immunology and food function. BMFH takes a multidisciplinary approach and focuses on a broad spectrum of issues.
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