Team:Technion Israel/Modifications/narx
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.
Fig. 1: Scheme of Tar chemoreceptor on the left, NarX-Tar chimera on the right
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.
As in the PctA, first a 3D model was generated using the website Phyre2, to insure the correct folding of all parts of the chimera.
The next step was to examine the activity of the receptor by conducting a chemotaxis assay. Since the NarX-Tar chimera is supposed to serve as a repellent chemoreceptor, the conventional assay is a chemical in plug assay, in which the repellent is added to a motility buffer and the movement of the bacteria is detected (See chemical in plug assay). Here sodium nitrate was used as a repellent for the NarX-Tar strain.
Later, a “Chip Microscope assay” was conducted. In this assay a suspension of the bacteria was confined into an “ibidi” microchannel chip and the bacterial concentration was monitored in a fixed point for the whole experiment. Later on, a solution of a repellent, sodium nitrate, in the concentrations of 10-2M and 10-6M was added to the channel. In addion, GFP was fused to the NarX-Tar chimera's C terminus in order to validate the location of the expressed chemoreceptor on the both poles of the bacterial membrane. The reporter protein was monitored using fluorescence microscopy followed by a flow cytometry.
Fig. 2: Biobrick device of the NarX-Tar chimera.
Results
The results of the 3D structure were not promising, as the folding was not achieved in the correct manner as can be seen in figure 1.
Fig. 1: NarX-Tar chimera 3D structure. The Tar signaling regions is in gray, the NarX LBD is in red.
When tested on the chemical in plug assay no movement was detected, indicating that the NarX-Tar strain is not responsive to sodium nitrate.
Fig. 2: Chemical in plug assay results: on the left, delta Z in the presence of the attractant Aspartic acid, on the right NarX-Tar strain with repellent sodium nitrate.
The expected results of the Chip Microscope assay were a decrease of the bacterial concentration overtime. Nevertheless, the strain showed no response to different concentrations of sodium nitrate, in other words it chimera did not function as expected. Lastly, the testing of the clone carrying the GFP gene fused to the chimera both with the flow cytometry and fluorescence microscopy showed no indication to fluorescence (add PDF link) .
Outlook
As the results showed that NarX-Tar strain failed to show any response to sodium nitrate. Moreover, as the reporter gene (GFP) showed no indication of expression, this subproject was put aside. It is unfortunate that the NarX-Tar clone was not successful, as it could have served as an additional proof of concept the S.Tar platform, in addition to the other strains we have cloned.
References:
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.
