Difference between revisions of "Team:Technion Israel/Proof"
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| − | One of S.Tars sub projects focused on altering | + | One of S.Tars sub-projects focused on altering the LBD of Tar chemoreceptor in order to design new hybrid chimeras. That was accomplished by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, without modifying Tar signaling region. As a proof of concept for the newly designed Tar chimeras and the S.Tar project, we focused on testing the PctA-Tar hybrid. |
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| − | PctA is a chemoreceptor found in the <i> Pseudomonas Aeruginosa </i> | + | PctA is a chemoreceptor found in the <i> Pseudomonas Aeruginosa </i>, it mediates |
chemotaxis towards amino acids and away from organic compounds. It can sense all | chemotaxis towards amino acids and away from organic compounds. It can sense all | ||
amino acids except for Aspartate <b>(1)</b>. | amino acids except for Aspartate <b>(1)</b>. | ||
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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" target="_blank">(K777000)</a>. Using these two sequences, we built a Biobrick device <a href="http://parts.igem.org/Part:BBa_K1992007" target="_blank">(K1992007)</a> | <a href="http://parts.igem.org/Part:BBa_K777000" target="_blank">(K777000)</a>. Using these two sequences, we built a Biobrick device <a href="http://parts.igem.org/Part:BBa_K1992007" target="_blank">(K1992007)</a> | ||
| − | + | wwhich was tested by transforming the device 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 (Parkinson J S, University of Utah)."> | data-content="An E.coli derivative, which lacks chemoreceptors genes, means this strain does not obtain chemotaxis ability (Parkinson J S, University of Utah)."> | ||
UU1250<i class="entypo-check"></i></button></a> | UU1250<i class="entypo-check"></i></button></a> | ||
| − | + | . It is important | |
to note that this chimera has been constructed before in the literature <b>(1)</b>. | to note that this chimera has been constructed before in the literature <b>(1)</b>. | ||
</p> | </p> | ||
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presented below, figure 4. It is important to mention that this assay was | 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 | performed on BA medium as the original assay on TB medium failed. From the | ||
| − | results seen | + | results seen in figure 5, it is |
| − | + | clear that the chimera functions and controls the chemotactic ability of | |
| − | the bacteria | + | the bacteria leading to swarming response. |
| + | This is compared to the control, UU1250 strain, that did not show any chemotactic ability as expected and no chemotactic rings were formed. | ||
| + | |||
</p> | </p> | ||
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<b>Fig. 5:</b> Swarming assay for attractant response of the PctA-Tar chimera. | <b>Fig. 5:</b> Swarming assay for attractant response of the PctA-Tar chimera. | ||
| − | <b>a.</b> PctA chimera, <b>b.</b> Negative control- UU1250 strain w/o the Tar expression plasmid, <b>c. </b> positive control - | + | <b>a.</b> PctA chimera, <b>b.</b> Negative control- UU1250 strain w/o the Tar expression plasmid, <b>c. </b> positive control - ΔZras strain expressing all chemoreceptors. |
<br><br><br> | <br><br><br> | ||
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| − | Next, to prove | + | Next, to prove that the chimera is localized in the membrane, GFP was fused |
to its C-terminus with a short linker sequence <a href="http://parts.igem.org/Part:BBa_K1992010" target="_blank">(K1992010)</a>, figure 6. The results of these tests | to its C-terminus with a short linker sequence <a href="http://parts.igem.org/Part:BBa_K1992010" target="_blank">(K1992010)</a>, figure 6. The results of these tests | ||
| − | as seen in figure 7, | + | as seen in figure 7, indeed show the expected localizations. |
</p> | </p> | ||
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| − | Finally, | + | Finally, in the video below is a working concept of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab project</a> - a chip that serves |
| − | as a detection tool based on the chemotaxis system of <I>E. coli</I> bacteria - | + | as a detection tool based on the chemotaxis system of <I>E. coli</I> bacteria - is presented.In the video a commercial ibidi microfluidic chip filled with a suspension of bacteria expressing the chimera and chromoprotein (<a href="http://parts.igem.org/Part:BBa_J23100" target="_blank">J23100</a> + <a href="http://parts.igem.org/Part:BBa_K1357009" target="_blank">K1357009</a>)can been seen. |
| − | + | A solution of repellent (10<sup>-3</sup>M Tetrachloroethylene) was added to the chip | |
| − | A solution of | + | |
and the displacement of the bacteria was monitored and recorded. | and the displacement of the bacteria was monitored and recorded. | ||
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<img src="https://static.igem.org/mediawiki/2016/thumb/9/95/T--Technion_Israel--FlashLab.png/800px-T--Technion_Israel--FlashLab.png" class="img-responsive img-center img-cont" style="cursor: pointer;"><br> </a> | <img src="https://static.igem.org/mediawiki/2016/thumb/9/95/T--Technion_Israel--FlashLab.png/800px-T--Technion_Israel--FlashLab.png" class="img-responsive img-center img-cont" style="cursor: pointer;"><br> </a> | ||
<p class="text-center"> | <p class="text-center"> | ||
| − | <b>Fig. 8:</b> A steps scheme of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> concept: Add bacteria | + | <b>Fig. 8:</b> A steps scheme of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> concept: Add bacteria expressing both the chemoreceptor of your choice and a chromoprotein to a fluidic chip. Add the tested sample to the chip. If the chemoreceptor detecets the substance in the sample, a displacement of the bacteria will become visible. |
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| − | In video 1, the displacement of the bacteria can be clearly seen in test chip (left chip, PctA-Tar with repellent), compared to | + | In video 1, the displacement of the bacteria can be clearly seen in test chip (left chip, PctA-Tar with repellent), compared to the control chip (right chip, PctA-Tar with buffer). |
</p> | </p> | ||
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With the supporting evidence of the results presented above, it can be concluded | With the supporting evidence of the results presented above, it can be concluded | ||
| − | that both concepts have been | + | that both concepts: LBD altering and the FlashLab platform, have been proven and work under real life conditions and seem promising for detection of various substances in the near future. |
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</p> | </p> | ||
</div> | </div> | ||
Revision as of 11:11, 19 October 2016
PctA-Tar chimera Introduction
One of S.Tars sub-projects focused on altering the LBD of Tar chemoreceptor in order to design new hybrid chimeras. That was accomplished by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, without modifying Tar signaling region. 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 , 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)
wwhich was tested by transforming the device to bacteria that lacks chemoreceptors -
UU1250
. 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 in figure 5, it is clear that the chimera functions and controls the chemotactic ability of the bacteria leading to swarming response. This is compared to the control, UU1250 strain, that did not show any chemotactic ability as expected and no chemotactic rings were formed.
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 - ΔZras strain expressing all chemoreceptors.
Next, to prove that the chimera is localized in the membrane, 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, indeed show the expected 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, in the video 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 - is presented.In the video a commercial ibidi microfluidic chip filled with a suspension of bacteria expressing the chimera and chromoprotein (J23100 + K1357009)can been seen. A solution of repellent (10-3M Tetrachloroethylene) 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 both the chemoreceptor of your choice and a chromoprotein to a fluidic chip. Add the tested sample to the chip. If the chemoreceptor detecets the substance in the sample, a displacement of the bacteria will become visible. .
In video 1, the displacement of the bacteria can be clearly seen in test chip (left chip, PctA-Tar with repellent), compared to the 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: LBD altering and the FlashLab platform, have been proven and work under real life conditions and seem promising for 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.
