ReViTA: A novel in vitro transcription system to study gene regulation

IF 4.5 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS New biotechnology Pub Date : 2023-09-25 DOI:10.1016/j.nbt.2023.04.005
Alba Rubio-Canalejas , Lucas Pedraz , Eduard Torrents
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Abstract

ReViTA (Reverse in Vitro Transcription Assay) is a novel in vitro transcription-based method to study gene expression under the regulation of specific transcription factors. The ReViTA system uses a plasmid with a control sequence, the promoter region of the studied gene, the transcription factor of interest, and an RNA polymerase saturated with σ70. The main objective of this study was to evaluate the method; thus, as a proof of concept, two different transcription factors were used, a transcriptional inducer, AlgR, and a repressor, LexA, from Pseudomonas aeruginosa. After the promoters were incubated with the transcription factors, the plasmid was transcribed into RNA and reverse transcribed to cDNA. Gene expression was measured using qRTPCR. Using the ReViTA plasmid, transcription induction of 55% was observed when AlgR protein was added and a 27% transcription reduction with the repressor LexA, compared with the samples without transcription factors. The results demonstrated the correct functioning of ReViTA as a novel method to study transcription factors and gene expression. Thus, ReViTA could be a rapid and accessible in vitro method to evaluate genes and regulators of various species.

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ReViTA:一种研究基因调控的新型体外转录系统
ReViTA(Reverse in Vitro Transcription Assay)是一种新的基于体外转录的方法,用于研究特定转录因子调控下的基因表达。ReViTA系统使用具有对照序列的质粒、所研究基因的启动子区、感兴趣的转录因子和饱和σ70的RNA聚合酶。本研究的主要目的是评估该方法;因此,作为概念的证明,使用了两种不同的转录因子,一种是来自铜绿假单胞菌的转录诱导因子AlgR,另一种是阻遏因子LexA。启动子与转录因子孵育后,将质粒转录为RNA并逆转录为cDNA。使用qRTPCR测量基因表达。使用ReViTA质粒,与没有转录因子的样品相比,当添加AlgR蛋白时观察到55%的转录诱导,并且用阻遏物LexA观察到27%的转录减少。结果证明了ReViTA作为一种研究转录因子和基因表达的新方法的正确功能。因此,ReViTA可能是一种快速且可获得的体外评估各种物种的基因和调节因子的方法。
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来源期刊
New biotechnology
New biotechnology 生物-生化研究方法
CiteScore
11.40
自引率
1.90%
发文量
77
审稿时长
1 months
期刊介绍: New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international. The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.
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