Difference between revisions of "Team:Technion Israel/Proof"
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Revision as of 22:04, 18 October 2016
PctA-Tar chimera Introduction
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
, 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).
Finally, demonstrated below in video 1 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. 8: 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.
In video 1, the displacement of the bacteria can be clearly seen in test chip (left chip, PctA-Tar with repellent), compared to he control chip (right chip, PctA-Tar with buffer).
Video 1: from left to right: (1) PctA-Tar chimera with Tetrachloroethylene repellent added. (2) PctA-Tar chimera with Motility buffer added (control).
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.
Histamine-Tar Introduction
The base of the S.Tar project is the Tar chemoreceptor, one of four E. coli receptors. Our goal is to create a engineered bacteria which has chemotaxis receptors sensetive to materials outside it’s existing receptor base.By changing Tar’s ligand binding domain (LBD) to that of other receptors from various sources or by mutating it we show that E. coli can be engineered to respond to completely new materials.
Fig. 1: Sequencing Results. Query describes the native Tar LBD sequence and Sbjct describes the design mutations sequence. Each mutation regin marked with another color (blue and red).
The bacterial world offers a relatively small selection of chemoreceptors in comparison to
the vast number of possible ligands. These receptors evolved specifically to recognize substances
which benefit or harm the organism in some way. On top of that the fact that the majority of known
receptors today are not well characterized meant that we had very few options of creating chimeric
receptors like we initially planned.
In light of the above we had to turn to a new path – redesigning the Tar chemoreceptor to bind a
different ligand using computational biology - The Rosetta software.
Out of the Rosetta’s 870 suggested mutations only 11 variants were eventually cloned into the native Tar ligan-binding domain LBD.
See Computational Design page for more information regarding the design process.
Out of all the tested variants only one was discovered to be attracted to histamine. Sequencing results
showed that the only mutations to occur in this variant were those planned by the Rosetta’s design.
Test and results
A microscopic observation was used order to test the bacteria’s response to the attractant, Histamine. It is evident in figure 1b that roughly 20 minutes after the addition of the Histamine, the concentration of bacteria in the vicinity of the Histamine is much greater than in the begining of the experiement as shown in figure 1a.
a.
b.
c.
d.
Fig. 2: microscope results of chemotaxis activity for variant His_9 with 10mM of Histamine. a. Tar-Histamine after 0 minutes (when the Histamine added). b. Tar-Histamine after 20 minutes. c. Control after 0 minutes (when the Histamine added). d. Control after 20 minutes.
To prove the correct localization of the LBD on both poles of the bacteria, GFP was fused to its C-terminus with a short linker sequence (E0040) . The results of these tests as seen in figure 2, prove our assumption of correct localizations.
Fig. 3: Results of GFP fusion. a. White light of Tar-Histamine-GFP b. Flourcense (490nm excitation) of Tar-Histamine-GFP c. White light of normal Tar d. Flourcense (490nm excitation) of normal Tar
Finally, demonstrated in video 1 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 chemoreceptor and chromoprotein (K1357008). A solution of Histamine in concentration of 10-3M, the attractant, was added to the chip and the displacement of the bacteria was monitored and recorded.
Video 1: from left to right:
(1) Histamine-Tar with Histamine atrractent added.
(2) Histamine-Tar with Motility buffer added (control).
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.
References:
1. Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513–525.
2. Moretti, R., Bender, B.J., Allison, B. and Meiler, J., 2016. Rosetta and the Design
of Ligand Binding Sites. Computational Design of Ligand Binding Proteins, pp.47-62.
