Difference between revisions of "Team:Technion Israel/Modifications/narx"

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in anaerobic respiration <b>(1)</b>. As a proof of concept of our platform, the Tar chemoreceptor LBD was replaced with  
 
in anaerobic respiration <b>(1)</b>. 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 <b>(1)</b>.  
 
the NarX LBD, using synthetic biology tools. A protocol which was previously shown to be successful has been recovered <b>(1)</b>.  
This work resulted in a NarX-Tar chimera, comprised by the NarX LBD and the C terminus of Tar chemoreceptor. The chimera  
+
This work resulted in a NarX-Tar chimera, comprised by the NarX LBD and Tar chemoreceptor's tail. The chimera  
 
was aimed to serve as a repellent chemoreceptor to nitrite and nitrate. </p>
 
was aimed to serve as a repellent chemoreceptor to nitrite and nitrate. </p>
 
</div>
 
</div>

Revision as of 07:41, 15 October 2016

S.tar, by iGEM Technion 2016

S.Tar, by iGEM Technion 2016


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 Tar chemoreceptor's tail. 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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Referances

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.



S.tar, by iGEM Technion 2016