Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones
Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones
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检测N-酰基高丝氨酸内酯的双荧光全细胞生物传感器的构建
DOI：
10.1016/s1001-0742(13)60407-6
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发表时间：
2014-02-01
影响因子：
6.9
通讯作者：
Zhuang, Xuliang
中科院分区：
文献类型：
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作者：
Deng, Xuemei;Zhuang, Guoqiang;Zhuang, Xuliang
N-酰基高丝氨酸内酯(AHLS)的检测有助于了解群体感应(QS)行为，包括生物膜的形成、毒力和代谢。为了原位检测AHL和指示宿主细胞，我们构建了pUCGMA2T(1-4)载体，构建了基于紫红色假单胞菌AHLI/R AHL系统的双荧光全细胞生物传感器。丁香属B728a。该质粒包含三个组成部分：结构性表达的P-nptII：指示宿主细胞的GFP，对AHL产生红色荧光的P-AhLi：：mCherry，以及编码AHL调节蛋白的ahlR基因。同时，T1-4的两个拷贝(四个串联拷贝。结果表明，该双荧光全细胞生物传感器能在6小时内对3OC6-HSL的5×10(-8)-1×10(-5)mol/L产生红色荧光。亮绿色荧光指示宿主细胞。此外，将该质粒转移到野生型假单胞菌PhTA125(一株产AHL的细菌)中，也显示出绿色和红色荧光。这一结果表明，该质粒可用于构建能够指示微环境中微生物的AHL反应和空间行为的全细胞指示剂。
Detection of N-acyl homoserine lactones (AHLs) is useful for understanding quorum sensing (QS) behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T(1-4) to make a dual fluorescent whole-cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components: constitutively expressed P-nptII:gfp for indicating host cells, P-ahlI::mcherry that produces red fluorescence in response to AHL, and the ahlR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 (four tandem copies of. a transcriptional terminator) were added into the plasmid to reduce background: The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence, whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 x 10(-8)-1 x 10(-5) mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wildtype Pseudomonas PhTA125 (an AHL-producing bacterium), it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a microenvironmental niche.