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用于光滑念珠菌的表达质粒。

Expression plasmids for use in Candida glabrata.

作者信息

Zordan Rebecca E, Ren Yuxia, Pan Shih-Jung, Rotondo Giuseppe, De Las Peñas Alejandro, Iluore Joseph, Cormack Brendan P

机构信息

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

出版信息

G3 (Bethesda). 2013 Oct 3;3(10):1675-86. doi: 10.1534/g3.113.006908.

Abstract

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.

摘要

我们描述了一系列含有不同光滑念珠菌启动子的CEN/ARS附加体质粒,这些质粒能够在光滑念珠菌中实现一系列蛋白质的组成型或调控型表达。启动子组包括三个组成型启动子(EGD2pr、HHT2pr、PDC1pr)、两个巨噬细胞/吞噬作用诱导型启动子(ACO2pr、LYS21pr)和一个营养调控型启动子(MET3pr)。每个启动子都被克隆到两个质粒骨架中,这两个骨架在其选择标记URA3或赋予对诺尔丝菌素耐药性的显性选择标记NAT1基因上有所不同。使用绿色荧光蛋白(GFP)作为报告基因,并通过流式细胞术或定量逆转录聚合酶链反应来评估表达水平,以此对所得到的12种质粒的表达情况进行评估。这组质粒共同扩展了可用于光滑念珠菌的表达载体工具集。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85e/3789792/4b83fcd96055/1675f1.jpg

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