Tools for characterization of Escherichia coli genes of unknown function
Tools for characterization of Escherichia coli genes of unknown function
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DOI：
10.1128/jb.184.16.4573-4581.2002
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发表时间：
2002-08-01
影响因子：
3.2
通讯作者：
Masters, M
中科院分区：
文献类型：
--
作者：
Merlin, C;McAteer, S;Masters, M
尽管测序和新兴高通量技术能够快速收集数据，但未知基因的明确功能表征仍然需要在个案研究中进行生化和遗传分析。这通常涉及靶基因的删除和删除物的表型表征。我们在此描述了对现有删除方法的修改，该方法通过用 FRT-lacZ-aph-P-lac-FRT 盒替换，可以促进删除过程，并能够方便地分析目标基因的表达特性。 lacZ 基因专门报告缺失基因的活性，因此可以确定其活跃表达的条件。编码卡那霉素抗性的 aph 基因提供了一种在菌株之间转导缺失基因座的可选择方法，以便该缺失可以与其他相关突变组合。 lac 启动子有助于克服操纵子内下游基因可能存在的极性效应。由于该盒的两侧是两个直接重复的 FRT 位点，因此可以通过反式提供的 Flp 重组酶将该盒切除。移除盒后会留下框内缺失，并伴有短灼烧，这不会干扰下游表达。使用盒制作的 yacF、yacG、yacH、yacK (cueO)、yacL、ruvA、ruvB、yabB 和 yabC 的替代品来验证其特性。
Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-lacZ-aph-P-lac-FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans. Removing the cassette leaves an in-frame deletion with a short sear which should not interfere with downstream expression. Replacements of yacF, yacG, yacH, yacK (cueO), yacL, ruvA, ruvB, yabB, and yabC made with the cassette were used to verify its properties.