Difference between revisions of "Team:Technion Israel/Proof"
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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>. | ||
| − | <br>To construct this chimera, the LBD sequence of the PctA was obtained from the <a href="http://www.pseudomonas.com/" | + | <br>To construct this chimera, the LBD sequence of the PctA was obtained from the <a href="http://www.pseudomonas.com/"><i> Pseudomonas </i> genome database</a>, |
| − | while the signaling region of Tar was obtained from the iGEM parts catalog <a href="http://parts.igem.org/Part:BBa_K777000" | + | 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> | ||
which was then transformed to bacteria that lacks chemoreceptors - <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" | 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."> | + | 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> |
| − | + | , to be extensively tested. 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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<div class="col-md-12 col-sm-12"> | <div class="col-md-12 col-sm-12"> | ||
<a class="pop ocenter"> | <a class="pop ocenter"> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/thumb/3/30/T--Technion_Israel--pctacircute.png/800px-T--Technion_Israel--pctacircute.png" class="img-responsive img-center" width="500 | + | <img src="https://static.igem.org/mediawiki/2016/thumb/3/30/T--Technion_Israel--pctacircute.png/800px-T--Technion_Israel--pctacircute.png" class="img-responsive img-center" width="500" style="cursor: pointer;"> |
</a> | </a> | ||
<p class="text-center"><b>Fig. 2:</b> Biobrick device of the PctA-Tar chimera.</p> | <p class="text-center"><b>Fig. 2:</b> Biobrick device of the PctA-Tar chimera.</p> | ||
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| − | <h2>Test and results | + | <h2>Test and results</h2> |
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<p class="text-justify"> | <p class="text-justify"> | ||
As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using the | As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using the | ||
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<a class="pop ocenter"> | <a class="pop ocenter"> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/6/62/T--Technion_Israel--Proof_fig3.png" class="img-responsive img-center img-cont" width="700 | + | <img src="https://static.igem.org/mediawiki/2016/6/62/T--Technion_Israel--Proof_fig3.png" class="img-responsive img-center img-cont" width="700" style="cursor: pointer;"> |
</a> | </a> | ||
<p class="text-center"><b>Fig. 3: </b> PctA-Tar chimera 3D structure. The Tar signaling regions is in gray, the PctA LBD is in red.</p> | <p class="text-center"><b>Fig. 3: </b> PctA-Tar chimera 3D structure. The Tar signaling regions is in gray, the PctA LBD is in red.</p> | ||
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Following the transformation, a swarming plate assay was performed in order to | 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 | confirm the functionality of the hybrid receptor. A scheme of the assay is | ||
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Next, to prove the correct localization of the chimera on both poles of the bacteria, GFP was fused | 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 < | + | 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, prove our assumption of correct localizations. | as seen in figure 7, prove our assumption of correct localizations. | ||
</p> | </p> | ||
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| − | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6e/T--Technion_Israel--pctaGFPcircute.png/800px-T--Technion_Israel--pctaGFPcircute.png" class="img-responsive img-center " width="500 | + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6e/T--Technion_Israel--pctaGFPcircute.png/800px-T--Technion_Israel--pctaGFPcircute.png" class="img-responsive img-center " width="500" style="cursor: pointer;"> |
</a> | </a> | ||
<p class="text-center"><b>Fig. 6:</b> Biobrick device of the PctA-Tar chimera fused to GFP.</p> | <p class="text-center"><b>Fig. 6:</b> Biobrick device of the PctA-Tar chimera fused to GFP.</p> | ||
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| + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6a/T--Technion_Israel--Tar_pctA_flourecent.png/800px-T--Technion_Israel--Tar_pctA_flourecent.png" class="img-responsive img-center img-cont" width="700" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 7:</b> Results of GFP fusion. <b>(A)</b> Positive control- <i>E.Coli</i> strain expressing GFP protein,<b>(B)</b> Negative control- UU1250 strain expressing Tar chemoreceptor, <b>(C)</b> UU1250 strain expressing Tar-GFP chemoreceptor, <b>(D)</b> UU1250 strain expressing PctA-Tar-GFP Chimera, Flourcense (490nm excitation). | ||
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Finally, demonstrated below is a working concept of the FlashLab project - a chip that serves | 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 <I>E. coli</I> bacteria - by using a commercial | as a detection tool based on the chemotaxis system of <I>E. coli</I> bacteria - by using a commercial | ||
| − | ibidi chip filled with a suspension of bacteria expressing the chimera and chromoprotein < | + | ibidi 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>). |
A solution of Tetrachloroethylene in concentration of 10<sup>-3</sup>M, the repellent, was added to the chip | A solution of Tetrachloroethylene in concentration of 10<sup>-3</sup>M, the repellent, was added to the chip | ||
and the displacement of the bacteria was monitored and recorded. | and the displacement of the bacteria was monitored and recorded. | ||
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| + | References:<br> | ||
| + | 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.<br> | ||
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Revision as of 10:32, 18 October 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
, 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 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.
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.
