Difference between revisions of "Team:Technion Israel/Proof"

Revision as of 10:06, 18 October 2016

S.tar, by iGEM Technion 2016

S.Tar, by iGEM Technion 2016

One of S.Tars sub projects focused on altering and changing the LBD of the Tar chemoreceptor in order to design new hybrid chimeras. These changes were made by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, while keeping the signaling region of Tar untouched. As a proof of concept for the newly designed Tar chimeras and the S.Tar project, we focused on testing the PctA-Tar hybrid.

Fig. 1: Scheme of native Tar chemoreceptor, native PctA receptor and PctA-Tar chimera. Adapted from (1)

PctA is a chemoreceptor found in the Pseudomonas Aeruginosa bacterium, it mediates chemotaxis towards amino acids and away from organic compounds. It can sense all amino acids except for Aspartate (1).
To construct this chimera, the LBD sequence of the PctA was obtained from the Pseudomonas genome database, while the signaling region of Tar was obtained from the iGEM parts catalog (K777000). Using these two sequences, we built a Biobrick device (K1992007) which was then transformed to bacteria that lacks chemoreceptors - UU1250, (Parkinson J S, University of Utah) to be extensively tested. It is important to note that this chimera has been constructed before in the literature (1).

Fig. 2: Biobrick device of the PctA-Tar chimera.

Test and results:

As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using the Phyre2 Protein Fold Recognition server to assure the correct folding of both the LBD and the signaling regions.

Fig. 3: PctA-Tar chimera 3D structure. The Tar signaling regions is in gray, the PctA LBD is in red.

Following the transformation, a swarming plate assay was performed in order to confirm the functionality of the hybrid receptor. A scheme of the assay is presented below, figure 4. It is important to mention that this assay was performed on BA medium as the original assay on TB medium failed. From the results seen below, figure 5, and compared to the negative control, it is clear that the chimera functions and controls the chemotactic ability of the bacteria and can lead to swarming response.

Fig. 4: a scheme for the swarming plate assay. In brief: Using a low percentage agar media plate, add a drop of bacteria to the middle of the plate. Incubate at 30 degrees for several hours. A halo or “chemotactic ring” should be formed in the agar plate.

a.

b.

c.

Fig. 5: Swarming assay for attractant response of the PctA-Tar chimera. a. PctA chimera, b. Negative control- UU1250 strain w/o the Tar expression plasmid, c. positive control - ΔZ strain expressing all chemoreceptors.

Next, to prove the correct localization of the chimera on both poles of the bacteria, GFP was fused to its C-terminus with a short linker sequence (K1992010), figure 6. The results of these tests as seen in figure 7, prove our assumption of correct localizations.

Fig. 6: Biobrick device of the PctA-Tar chimera fused to GFP.

Fig. 7: Results of GFP fusion. (A) Positive control- E.Coli strain expressing GFP protein,(B) Negative control- UU1250 strain expressing Tar chemoreceptor, (C) UU1250 strain expressing Tar-GFP chemoreceptor, (D) UU1250 strain expressing PctA-Tar-GFP Chimera, Flourcense (490nm excitation).

Fig. 8: =========fill here!!!=====

Finally, demonstrated below is a working concept of the FlashLab project - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria - by using a commercial ibidi chip filled with a suspension of bacteria expressing the chimera and chromoprotein (J23100 + K1357009). A solution of Tetrachloroethylene in concentration of 10^-3M, the repellent, was added to the chip and the displacement of the bacteria was monitored and recorded.

Fig. 9: A steps scheme of the FlashLab concept: Add bacteria expressing the chemoreceptor of your choice and a chromo protein to a fluidic chip . Add the sample in question to said chip. If the sample contains the substance that is recognized by the chemoreceptor, a displacement of the bacteria will become visible. If not, then the no displacement will be seen.

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With the supporting evidence of the results presented above, it can be concluded that both concepts have been proved and work under real life conditions and might lead to the detection of various substances in the near future.

@@ Line 176: / Line 176: @@
 													amino acids except for Aspartate <b>(1)</b>.
 													<br>To construct this chimera, the LBD sequence of the PctA was obtained from the <a href="http://www.pseudomonas.com/"><b><i> Pseudomonas </i> genome database</b></a>,
-													 while the signaling region of Tar was obtained from the iGEM parts catalog
+													 while the signaling region of Tar was obtained from the iGEM parts catalog <a href="http://parts.igem.org/Part:BBa_K777000" ><b>(K777000)</b></a>. Using these two sequences, we built a Biobrick device <a href="http://parts.igem.org/Part:BBa_K1992007" ><b>(K1992007)</b></a>
-													<b>(K777000)</b>. Using these two sequences, we built a Biobrick device <b>(K1992007)</b>
 													which was then transformed to bacteria that lacks chemoreceptors - <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
 															data-content="An E.coli derivative, which lacks chemoreceptors genes, means this strain does not obtain chemotaxis ability.">