A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments |
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| Affiliation: | 1. Department of Gastroenterology, Ajou University School of Medicine, Suwon, Republic of Korea;2. Department of Radiology, Ajou University School of Medicine, Suwon, Republic of Korea;3. Department of Surgery, Ajou University School of Medicine, Suwon, Republic of Korea;1. School of Computer Science and Technology, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong, China;2. Key Laboratory of Network Oriented Intelligent Computation, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong, China;1. Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Cologne, Germany;2. Department of Ophthalmology, University Hospital Regensburg, Regensburg, Germany;3. Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Regensburg, Germany;4. Institute for Medical Microbiology, Immunology and Hygiene and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany;1. Central Laboratory, the Second Hospital of Jilin University, Key Laboratory of Zoonosis research, Ministry of Education, Changchun 130041, China;2. College of Life and Ocean Science, Shenzhen University, Shenzhen 518060, China;1. Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark;2. Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark;3. Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark;1. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Equipe 12, F-75006, Paris, France;2. INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France;3. Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France |
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| Abstract: | The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize Gram- bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of approx. 109 bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177–250 μm) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 105 cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria. |
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