Wegener Marius, Vogtmann Kristina, Huber Madeleine, Laass Sebastian, Soppa Jörg
Goethe-University, Biocentre, Institute for Molecular Biosciences, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
J Microbiol Methods. 2016 Dec;131:181-187. doi: 10.1016/j.mimet.2016.10.015. Epub 2016 Oct 26.
Reporter genes facilitate the characterization of promoter activities, transcript stabilities, translational efficiencies, or intracellular localization. Various reporter genes for Escherichia coli have been established, however, most of them have drawbacks like transcript instability or the inability to be used in genetic selections. Therefore, the glpD gene encoding glycerol-3-phosphate dehydrogenase was introduced as a novel reporter gene for E. coli. The enzymatic assay was optimized, and it was verified that growth on glycerol strictly depends on the presence of GlpD. The 5'-UTRs of three E. coli genes were chosen and cloned upstream of the new reporter gene glpD as well as the established reporter genes lacZ and gusA. Protein and transcript levels were quantified and translational efficiencies were calculated. The lacZ transcript was very unstable and its level highly depended on its translation, compromising its use as a reporter. The results obtained with gusA and glpD were similar, however, only glpD can be used for genetic selections. Therefore, glpD was found to be a superior novel reporter gene compared to the established reporter genes lacZ and gusA.
报告基因有助于启动子活性、转录稳定性、翻译效率或细胞内定位的表征。已经建立了多种用于大肠杆菌的报告基因,然而,其中大多数都存在缺点,如转录不稳定或无法用于遗传筛选。因此,引入了编码甘油-3-磷酸脱氢酶的glpD基因作为大肠杆菌的新型报告基因。对酶活性测定进行了优化,并证实了在甘油上的生长严格依赖于GlpD的存在。选择了三个大肠杆菌基因的5'-非翻译区,并将其克隆到新报告基因glpD以及已建立的报告基因lacZ和gusA的上游。对蛋白质和转录水平进行了定量,并计算了翻译效率。lacZ转录本非常不稳定,其水平高度依赖于其翻译,这影响了它作为报告基因的用途。用gusA和glpD获得的结果相似,然而,只有glpD可用于遗传筛选。因此,与已建立的报告基因lacZ和gusA相比,发现glpD是一种更优越的新型报告基因。