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A uropathogenic Escherichia coli (UPEC) strain for intravital studies of an infection

In our model we study UPEC infecting a proximal tubule in a rat kidney. In order to be able to follow the infecting bacterium under the microscope, when inside the tissue, the bacterium needs to express a fluorescent substance. We chose green fluorescent protein (GFP). Plasmids expressing GFP are available, but they are not a practical solution in these experiments since no selection for the plasmid can be applied. Therefore we chose to insert a single copy of the gfp-gene into the chromosome by the method described by Datsenko and Wanner 2000. As insertion position on the chromosome we chose a gene that codes for a protein with no known function E coli. In Salmonella CobS is part of cobalamin synthesis, not active in E coli.

The commonly used promoter PrpsM could not be used in these studies as it governs the synthesis of a ribosomal protein. Expression of ribosomal components are known to respond to changes in environment and we needed expression to be constant under all different conditions. Therefore, we chose PLtetO-1, which in the abscence of the repressor protein TetR becomes constitutive (Bertram and Hillen 2008).

GFP

The figure shows the linear DNA that was inserted into the cobS gene. First is the promoter followed by the gfp gene. At the end is a gene coding for chloramphenicol resistance serving as a selective marker.

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