Korpela M T, Kurittu J S, Karvinen J T, Karp M T
Department of Biotechnology, University of Turku, Finland.
Anal Chem. 1998 Nov 1;70(21):4457-62. doi: 10.1021/ac980740e.
A bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline group of antibiotics is described. A sensor plasmid, containing five genes from bacterial luciferase operon of Photorhabdus luminescens inserted under the control of tetracycline-responsive elements of the transposon Tn10, was constructed. Usage of the full-length luciferase operon in the sensor resulted in tetracycline-dependent light production without additions, i.e., self-luminescent phenotype, since all the substrates were intrinsically produced by the recombinant organism. The time needed for optimal induction of light emission was 90 min. Maximal induction of approximately 100-fold over uninduced levels by using 20 ng of tetracycline, and picomole sensitivities for the seven different tetracyclines tested, were obtained without added Mg2+ ions. The higher the pH and the magnesium ion concentration in the assay medium the higher was the amount of membrane-impermeable tetracycline-Mg2+ chelate complex. In consequence, by adjusting the pH and the Mg2+ ion concentration, the sensitivity of the assay can be modified for different analytical purposes. Different non-tetracycline antibiotics did not cause induction of light emission.
描述了一种用于特异性检测四环素类抗生素的生物发光大肠杆菌K-12菌株。构建了一种传感器质粒，其含有来自发光杆菌属细菌荧光素酶操纵子的五个基因，插入在转座子Tn10的四环素响应元件的控制下。由于所有底物均由重组生物体固有产生，因此传感器中全长荧光素酶操纵子的使用导致在不添加任何物质的情况下产生四环素依赖性发光，即自发光表型。最佳诱导发光所需的时间为90分钟。在不添加Mg2+离子的情况下，使用20 ng四环素可实现比未诱导水平最大约100倍的诱导，并且对所测试的七种不同四环素具有皮摩尔灵敏度。测定培养基中的pH值和镁离子浓度越高，膜不可渗透的四环素-Mg2+螯合物的量就越高。因此，通过调节pH值和Mg2+离子浓度，可以针对不同的分析目的改变测定的灵敏度。不同的非四环素类抗生素不会引起发光诱导。
