Team:Technion Israel/Proof
Headline
Small Headline
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: Tar chemoreceptor on the left, PctA-Tar chimera on the right. (source@#$%^&*)
PctA is a chemoreceptor found in the Pseudomonas Aeruginosa bacterium, it mediates chemotaxis toward
amino acids and away from organic compounds. It can sense all amino acids except for Aspartate. (1).
To construct this chimera, the LBD sequence of the PctA was obtained from the Pseudomonas genome database
while the signaling region of Tar was obtained from the iGEM parts catalog (part number). Using these two
sequences, we built a Biobrick device (part number) which was then transformed to bacteria that lacks
chemoreceptors - UU1250 (Parkinson J S, University of Utah) to be extensively tested. It is important
to note that this chimera has been constructed before in the literature (1).
Fig. 2: Biobrick device of the PctA-Tar chimera. (source@#$%^&*)
Test and results:
As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using Phyre2 website 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. (source@#$%^&*)
Following the transformation, a swarming plate assay was performed in order to confirm the functionality of the hybrid receptor. It is important to mention that this assay was performed on BA medium as the original assay on TB medium failed. From the results seen below, figure 2, and compared to the negative control, it is clear that the chimera functions and controls the chemotactic ability of the bacteria and can lead to swarming response.
a.
b.
c.
Fig. 5: ??? Results of swarming assay of (a) PctA-Tar chimera, (b) Negative control (UU1250) (c) Positive control (B275 ∆Zras).
Next, to prove the correct localization of the chimera on both poles of the bacteria, GFP was fused to its C-terminus with the a short linker sequence (part number). The results of these tests as seen in figure 3, prove our assumption of correct localizations.
Fig. 6: Biobrick device of the PctA-Tar chimera fused to GFP. (source@#$%^&*)
a.
b.
c.
d.
Fig. 7: ??? Results of GFP fusion. (a) White light of PctA-Tar-GFP Chimera (b) Flourcense (490nm excitation) of PctA-Tar-GFP Chimera (c) White light of normal Tar (d) Flourcense (490nm excitation) of normal Tar.
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 ibd chip filled with a suspension of bacteria expressing the chimera and chromoprotein (part number). A solution of Tetrachloroethylene in concentration of 9×10-5M, the repellent, was added to the chip and the displacement of the bacteria was monitored and recorded.
Fig. 8: A steps scheme of the FlashLab concept. To a fluidic chip add bacteria expressing the chemoreceptor of your choice and a chromo protein. Add the sample in question to said chip. If the sample contains the substance that is recognized by the chemoreceptor, then displacement of the bacteria will be visible. If not, then the no displacement will be seen. (source@#$%^&*)
MISSING VIDEO
To conclude, all results presented above support the proof of concept, as we were able to see the movement of bacteria due to the chemotaxis and thus being able to detect different compounds.
1. ???
2. ???
3. ???
