Difference between revisions of "Team:Technion Israel/Modifications/narx"
| Line 247: | Line 247: | ||
To test the chemotactic ability of the cloned strain chemotaxis assays were performed. | To test the chemotactic ability of the cloned strain chemotaxis assays were performed. | ||
<br>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 <a href="https://2016.igem.org/Team:Technion_Israel/Experiments">chemical in plug assay</a>). Here sodium nitrate was used as a repellent for the NarX-Tar strain. | <br>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 <a href="https://2016.igem.org/Team:Technion_Israel/Experiments">chemical in plug assay</a>). Here sodium nitrate was used as a repellent for the NarX-Tar strain. | ||
| − | <br>Later, a <a href="https://static.igem.org/mediawiki/2016/f/f3/T--Technion_Israel--Chemotaxis_assays.pdf" target="_blank">drop assay</a> was conducted. A suspension of bacteria in motility buffer was placed on a microscope slide and a drop of repellent was added in the concentrations of 10- | + | <br>Later, a <a href="https://static.igem.org/mediawiki/2016/f/f3/T--Technion_Israel--Chemotaxis_assays.pdf" target="_blank">drop assay</a> was conducted. A suspension of bacteria in motility buffer was placed on a microscope slide and a drop of repellent was added in the concentrations of 10<sup>-2</sup>M and 10<sup>-6</sup>M. The slide was monitored throughout the entire procedure in order to examine the concentration change of bacteria in the drop. |
<br>In addion, GFP was fused to the NarX-Tar chimera's C terminus, in order to validate the location of the expressed chemoreceptor on both poles of the bacterial membrane. The reporter protein was monitored using fluorescence microscopy followed by a flow cytometry. | <br>In addion, GFP was fused to the NarX-Tar chimera's C terminus, in order to validate the location of the expressed chemoreceptor on both poles of the bacterial membrane. The reporter protein was monitored using fluorescence microscopy followed by a flow cytometry. | ||
Revision as of 12:17, 19 October 2016
Introduction
As a proof of concept of our platform, the Tar chemoreceptor LBD was replaced with the NarX LBD. The NarX sensor protein of E. coli binds nitrite and nitrate and induces the expression of proteins involved in anaerobic respiration (1). We followed A a protocol which was previously shown to be successful (1). This work resulted in a NarX-Tar chimera, comprised by the NarX LBD and Tar’s cytoplasmic region. According to the article 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 construct this chimera, the protein sequence of both the LBD and the linker region of NarX were obtained the literature (1).
Using the DNA sequences of both frgamnets and Tar’s cytoplasmic region, we built a Biobrick device (part not submitted) (2). As in the PctA, first a 3D model was generated using the website Phyre2, to ensure the correct folding of all parts of the chimera (Figure 1, results).
The next step was transforming to bacteria that lacks chemoreceptors - UU1250 (Parkinson J S, University of Utah).
To test the chemotactic ability of the cloned strain chemotaxis assays were performed.
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 drop assay was conducted. A suspension of bacteria in motility buffer was placed on a microscope slide and a drop of repellent was added in the concentrations of 10-2M and 10-6M. The slide was monitored throughout the entire procedure in order to examine the concentration change of bacteria in the drop.
In addion, GFP was fused to the NarX-Tar chimera's C terminus, in order to validate the location of the expressed chemoreceptor on 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 1a. The 3D structure of the native Tar is presented in figure 1b.
a.
b.
Fig. 1: (a)NarX-Tar chimera 3D structure. The Tar signaling regions is in gray, the NarX LBD is in red. (b)Native Tar 3D structure.
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 drop assay were a decrease of the bacterial
concentration overtime. Nevertheless, the strain showed no response to different concentrations of sodium nitrate. Unfortunately a sufficient documentation hasn't been obtained.
In addition, an attempt to fuse GFP to NarX-Tar chimera has failed. Lastly, the testing of the clone carrying the GFP gene fused to the chimera with flow cytometry showed no indication to fluorescence. See results here.
Outlook
As the results showed, 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.
