Team:Technion Israel/Modifications/narx
NarX-Tar - Introduction
Introduction
The NarX sensor of E. coli mediates response to nitrite and nitrate, which results in gene expression that are involved in anaerobic respiration (1). As a proof of concept of our platform, the Tar chemoreceptor LBD was replaced with the NarX LBD, using synthetic biology tools. 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 the C terminus of Tar chemoreceptor. The chimera was aimed to serve as a repellent chemoreceptor to nitrite and nitrate.
Design and Implementation
The codon for the required fragments, which are the NarX LBD and the linker rigion were obtained from the literature (1).
The sequence of the desired segments for both the NarX and Tar’s cytoplasmic region were obtained from the complete E coli genome sequence (2)
The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah) and a
chemotaxis assay on chip under a microscope was conducted using different concentrations of sodium nitrate as a repellent.
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.
