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Revision as of 09:54, 16 October 2016
Introduction
The NarX sensor protein of E. coli binds nitrite and nitrate and induces the expression of proteins involved in anaerobic respiration (1). As a proof of concept of our platform, the Tar chemoreceptor LBD was replaced with the NarX LBD. A protocol which was previously shown to be successful has been recovered (1). This work resulted in a NarX-Tar chimera, comprised by the NarX LBD and Tar’s cytoplasmic region. The chimera was supposed to function as a repellent chemoreceptor to nitrite and nitrate.
Design and Implementation
To produce this chimera, the protein sequence of both the LBD and the linker region of NarX were obtained from the literature (1).
Using the DNA sequences of both mentioned segments and Tar’s cytoplasmic region, that were obtained from the complete E. coli genome sequence (2)
we built a Biobrick device (part not submitted) which was then transformed to bacteria that lacks chemoreceptors - UU1250 (Parkinson J S, University of Utah). The transformation was followed by different assays in order to test the chemotactic ability of the chimera.
The bacteria were confined into an ibidi mifrochannel and the sodium nitrate was inserted through the well in the concentrations
of 10-2M and 10-6M. The chip was monitored throughout the entire procedure in order to examine the
concentration change of bacteria next to the well.
Furthermore, a GFP gene was fused to the NarX-Tar chimera's C terminus in order to validate the location of the expressed chemoreceptor
on the bacterial membrane. The fusion location was monitored using fluorescence microscopy followed by a FACS test for fluorescence.
Results
During the chemotaxis assay on chip under the microscope, the concentration the bacteria adjacent to the well, was expected
to decrease through time. Nevertheless, the strain showed no response to different concentrations of sodium nitrate.
When testing the clone carrying the GFP fused to the chimera both the FACS and fluorescence microscopy and the FACS showed
no indication to fluorescence, probably due to a problem in the expression process.
Outlook
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1. R Ward, S.M., Delgado, A., Gunsalus, R.P., and Manson, M.D. (2002). A NarX-Tar chimera mediates repellent chemotaxis to nitrate and nitrite. Mol. Microbiol. 44, 709–719.
2. The complete E coli genome sequence.
