Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.
Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.
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DOI：
10.1016/bs.mie.2017.03.013
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发表时间：
2017
影响因子：
--
通讯作者：
Camps M
中科院分区：
文献类型：
--
作者：
Standley M;Allen J;Cervantes L;Lilly J;Camps M
模式生物暴露于化学品后发生的突变被用作遗传毒性的指标。诱变试验也用于研究DNA稳态的机制。本文主要介绍了原核生物突变检测的两种方法：报告基因失活（正向突变试验）和失活突变的逆转（逆转突变试验）。这两种方法都是劳动密集型的，包括目视筛选，固体培养基上菌落的定量，或确定液体培养中的泊松分布。在这里，我们提出了两个体内诱变的反转报告，产生定量输出，因此有可能大大减少测试化学物质的数量和劳动力参与这些分析。通过将两个基因放在同一质粒上，或者通过翻译融合并用终止密码子中断ORF的n端，将基于TEM β内酰胺酶的还原实验与GFP荧光相结合，获得了该输出。我们还描述了一个旨在促进监测突变株连续诱变的报告器。该报告基因偶联了两个逆转标记，允许突变事件在时间上的时间分离，从而提供了关于突变菌株突变动力学的信息。在这里，我们描述了这些报告系统，提供了使用协议，并使用易出错的Pol I突变作为突变源展示了它们的关键功能特征。
Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations.
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影响因子：
5.3
作者：
Marinus, Martin G.
通讯作者：
Marinus, Martin G.
DOI：
10.1080/10937404.2010.483176
发表时间：
2010-02
期刊：
Journal of toxicology and environmental health. Part B, Critical reviews
影响因子：
--
作者：
Krewski D;Acosta D Jr;Andersen M;Anderson H;Bailar JC 3rd;Boekelheide K;Brent R;Charnley G;Cheung VG;Green S Jr;Kelsey KT;Kerkvliet NI;Li AA;McCray L;Meyer O;Patterson RD;Pennie W;Scala RA;Solomon GM;Stephens M;Yager J;Zeise L
通讯作者：
Zeise L
DOI：
10.1073/pnas.1210309109
发表时间：
2012-10-09
影响因子：
11.1
作者：
Lee, Heewook;Popodi, Ellen;Foster, Patricia L.
通讯作者：
Foster, Patricia L.
影响因子：
16.6
作者：
Badran AH;Liu DR
通讯作者：
Liu DR
DOI：
10.1007/978-1-4939-1053-3_3
发表时间：
2014-01-01
期刊：
DIRECTED EVOLUTION LIBRARY CREATION: METHODS AND PROTOCOLS, 2ND EDITION
影响因子：
--
作者：
Alexander, David L.;Lilly, Joshua;Camps, Manel
通讯作者：
Camps, Manel