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

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<p class="text-justify">
 
<p class="text-justify">
−
We successfully redesigned, clone and test a novel Histamine-Tar chemoreceptor.
+
We successfully redesigned, clone and test a novel Histamine-Tar  
−
The design made with Rosetta bioinformatics tool, by following a protocol presented  
+
chemoreceptor. The design made with Rosetta bioinformatics tool,  
−
in "Rosetta and the Design of Ligand Binding Sites" (1), in order  
+
by following a protocol presented in "Rosetta and the Design of  
−
to design a binding site around a selected small molecule ligand.  
+
Ligand Binding Sites" (1), in order to design a binding site around a  
−
The Rosetta’s design process for the new ligand Histamine produced 870 results, out of which 11 variants
+
selected small molecule ligand. The Rosetta’s design process for
−
remained after filtering. The 11 variants were cloned into the native Tar  
+
the new ligand Histamine produced 870 results, out of which 11  
−
ligand-binding domain(LBD), out of them only 6 exhibited the expected  
+
variants remained after filtering. The 11 variants were cloned  
−
sequences in sequencing and were subjected to chemotaxis tests.  
+
into the native Tar ligand-binding domain(LBD), out of them only  
−
Out of 6 tested variants only one of Tar mutated variants (variant number 9) was  
+
6 exhibited the expected sequences in sequencing and were  
−
successfully designed and verified to bind Histamine as a ligand, instead of Aspartate.
+
subjected to chemotaxis tests. Out of all the tested variants  
−
The results of the chemotaxis test for variant number 9 are presented in figure 2.
+
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.
 
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</p>
 
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diffrent color (blue and red).</p>
 
diffrent color (blue and red).</p>
 
</div>
 
</div>
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</div>
 +
 +
<br>
 +
<!-- 12 text div -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<p class="text-justify">
 +
We observed the bacteria’s response to the attractant,
 +
Histamine, by using a microscope. It is evident in figure
 +
2b that roughly 20 minutes after the addition of the Histamine,
 +
the bacteria concentration in the vicinity of the Histamine is
 +
much greater than in the the beginning of the experiment (figure 2a).
 +
</p>
 +
</div>
 
</div>
 
</div>
 
<br><br>
 
<br><br>
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<p class="text-center"><b>Fig. 2:</b>  microscope results of chemotaxis activity for variant His_9 with 10mM of Histamine.<b> a.</b> Tar-Histamine after 0 minutes (when the Histamine added).<b> b.</b> Tar-Histamine after 20 minutes.<b> c.</b> Control after 0 minutes (when the Histamine added).<b> d.</b> Control after 20 minutes.
+
<p class="text-center"><b>Fig. 2:</b>   
 +
microscope results of chemotaxis activity for variant His_9 with 10mM Histamine.
 +
<b>a.</b> Tar-Histamine: before adding Histamine). <b>b.</b> Tar-Histamine:
 +
20 minutes after adding Histamine. <b>c.</b> Tar-Histamine: before adding the
 +
Motility buffer (control solution). <b>d.</b> Tar-Histamine: 20 minutes after
 +
adding the Motility buffer.
 
</p>
 
</p>
 
</div>
 
</div>
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<br><br>
 
<br><br>
 +
 +
 +
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<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<h4>Histamine-Tar fused with GFP marker:</h4>
 +
<p class="text-justify">
 +
To prove the correct localization of the Tar-Histamine,
 +
GFP was fused to its C-terminus with a short linker sequence <a href="http://parts.igem.org/Part:BBa_E0040" target="_blank">(E0040)</a>.
 +
The results of these tests as seen in figure 3, prove our assumption
 +
of correct localizations.
 +
</p>
 +
</div>
 +
</div>
 +
 +
<!-- 12 img div -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<a class="pop ocenter">
 +
<img src="https://static.igem.org/mediawiki/2016/thumb/e/e3/T--Technion_Israel--His-GFP.png/800px-T--Technion_Israel--His-GFP.png" class="img-responsive img-center img-cont" width="450" style="cursor: pointer;">
 +
</a>
 +
<p class="text-center"><b>Fig. 3:</b>
 +
Results of GFP fusion. <b>a.</b> Positive control- E.Coli 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 Histamine-Tar-GFP Chimera,
 +
fluorescence (490nm excitation).
 +
</p>
 +
</div>
 +
</div>
 +
 +
<div class="row">
 +
<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
 +
<p class="text-justify">
 +
Finally, in the video below, 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 E.
 +
coli bacteria – is presented. In the video a commercial ibidi microfluidic chip filled with a suspension of
 +
bacteria expressing the chemoreceptor and chromoprotein
 +
(<a href="http://parts.igem.org/Part:BBa_K1992011" target="_blank">K1992011</a> + <a href="http://parts.igem.org/Part:BBa_K1357008" target="_blank">K1357008</a>) can been seen.
 +
A solution of attractant (10<sup>-3</sup>M Histamine)
 +
was added to the chip and bacteria displacement was monitored and recorded
 +
</p>
 +
</div><!--
 +
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
 +
<a class="pop ocenter">
 +
<img src="https://static.igem.org/mediawiki/2016/7/74/T--Technion_Israel--His_control.gif" class="img-responsive img-center img-cont" width="150" style="cursor: pointer;">
 +
</a>
 +
<br>
 +
<!--video autoplay loop class="embed-responsive-item video-size img-cont">
 +
<source src="https://static.igem.org/mediawiki/2016/4/45/T--Technion_Israel--PctA_and_His.mp4" type="video/mp4">
 +
</video-->
 +
<p class="text-center"><b>Video 1:</b> from left to right:
 +
(1) Histamine-Tar with Histamine attractant added.
 +
(2) Histamine-Tar with Motility buffer added (control).<br>
 +
</p>
 +
</div>
 +
<br><br><br>
 +
</div>
 +
 
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<h4>PctA-Tar fused with GFP marker:</h4>
 
<h4>PctA-Tar fused with GFP marker:</h4>
 
<p class="text-justify">
 
<p class="text-justify">
−
In addition, the correct localization of the chimera on both poles of the bacteria  
+
In addition, the correct localization of the chimera on both  
−
was proven through fusion of GFP to the C- terminus of the chimera. The results of  
+
poles of the bacteria membrane, was proven through fusion of  
−
these tests as seen in figure 4, prove the correct localizations.
+
GFP to the C- terminus of the chimera with a short linker
 +
sequence <a href="http://parts.igem.org/Part:BBa_K1992010" target="_blank">(K1992010)</a>. The results of these tests as seen in figure 4,  
 +
indeed show the expected localizations.
 
</p>
 
</p>
 
</div>
 
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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;">
 
<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>
 
</a>
−
<p><b>Fig. 4:</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).
+
<p><b>Fig. 4:</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).
 
</p>
 
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<br>
 
<br>
 
<br>
 
<br>
 +
 +
<!-- 12 text div -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<p class="text-justify">
 +
Finally, in the video above, video 2, 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 E. coli 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
 +
and the displacement of the bacteria was monitored and recorded.
 +
</p>
 +
</div>
 +
</div>
  
 +
 +
<!-- Mini headline -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<h2>Trap and Track - Novel assay for digital chemotaxis detection</h2>
 +
</div>
 +
</div>
  
 +
<!-- 12 text div -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<p class="text-justify">
 +
Although there is an abundant number of chemotaxis assays available
 +
today, most of them were designed 40 to 50 years ago and almost
 +
none provide a real time measurement without the use of fluorescence labeling.<br>
 +
The use of Porous Si (PSi) and oxidized PSi (PSiO2) matrices for biological
 +
sensing is on the rise. So far various analytes such as DNA, proteins and
 +
bacteria have been proven to be detectable on such matrices. The common
 +
method to monitor the interaction of said analytes within the porous films
 +
is reflective interferometric Fourier transform spectroscopy (RIFTS), as it
 +
allows a real time measurement and output for the user.<br>
 +
We present the results of an early experiment for the detection of
 +
chemotactic activity on the porous silicon films initially developed for bacterial detection.<br>
 +
<br>
 +
This assay provides us with the means to digitally quantify chemotaxis -
 +
meaning we have a clear distinction between an attractant response
 +
(negative trend), repellent response (positive trend) and no response (zero trend).<br>
 +
<br>
 +
The following graph presents only the chemotactic responses taken from 4
 +
different experiments, The top 2 plots, in blue and in grey, represent a
 +
repellent response. The yellow plot represents a negative control experiment
 +
and the bottom red graph represents an attractant response.<br>
 +
For more information see our <a href="https://2016.igem.org/Team:Technion_Israel/Measurement">Measurement page</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 +
<!-- 12 img div -->
 +
<div class="row">
 +
<div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
 +
 +
<a class="pop ocenter">
 +
<img src="https://static.igem.org/mediawiki/2016/8/86/T--Technion_Israel--Measurement9.png" class="img-responsive img-center img-cont" width="450" style="cursor: pointer;">
 +
</a>
 +
<p class="text-center"><b>Fig. 5:</b>Digital chemotaxis measurements.</p>
 +
</div>
 +
</div>
  
 
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Revision as of 15:34, 19 October 2016

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 2016

Histamine-Tar novel chemoreceptor

We successfully redesigned, clone and test a novel Histamine-Tar chemoreceptor. The design made with Rosetta bioinformatics tool, by following a protocol presented in "Rosetta and the Design of Ligand Binding Sites" (1), in order to design a binding site around a selected small molecule ligand. The Rosetta’s design process for the new ligand Histamine produced 870 results, out of which 11 variants remained after filtering. The 11 variants were cloned into the native Tar ligand-binding domain(LBD), out of them only 6 exhibited the expected sequences in sequencing and were subjected to chemotaxis tests. 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.

Fig. 1: Sequencing Results. Query describes the native Tar LBD sequence and Sbjct describes the design mutations sequence. Each mutation regin marked with diffrent color (blue and red).


We observed the bacteria’s response to the attractant, Histamine, by using a microscope. It is evident in figure 2b that roughly 20 minutes after the addition of the Histamine, the bacteria concentration in the vicinity of the Histamine is much greater than in the the beginning of the experiment (figure 2a).



a.

b.

c.

d.

Fig. 2: microscope results of chemotaxis activity for variant His_9 with 10mM Histamine. a. Tar-Histamine: before adding Histamine). b. Tar-Histamine: 20 minutes after adding Histamine. c. Tar-Histamine: before adding the Motility buffer (control solution). d. Tar-Histamine: 20 minutes after adding the Motility buffer.



Histamine-Tar fused with GFP marker:

To prove the correct localization of the Tar-Histamine, GFP was fused to its C-terminus with a short linker sequence (E0040). The results of these tests as seen in figure 3, prove our assumption of correct localizations.

Fig. 3: 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 Histamine-Tar-GFP Chimera, fluorescence (490nm excitation).

Finally, in the video below, 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 chemoreceptor and chromoprotein (K1992011 + K1357008) can been seen. A solution of attractant (10-3M Histamine) was added to the chip and bacteria displacement was monitored and recorded


Video 1: from left to right: (1) Histamine-Tar with Histamine attractant added. (2) Histamine-Tar with Motility buffer added (control).




As we showed, Rosetta provides us with the means to redesign a chemoreceptor to bind new ligands. In the future this ability can be used in the same manner to design dozens of new receptors. The critical step of the design process remains the lab work required to clone and test the variants, this step can be optimized by using a high throughput chemotaxis assay. Aside from this, any receptor designed can be further improved by introducing a directed evolution step to improve its specificity towards the new ligand.

In addition, we wrote “How to use” Rosetta guide with Rosetta developers and iGEM TU Eindhoven team, and build a framwork software tool to allow fellow researchers the ability to redesign a binding site easlly.

FlashLab - microfluidic chip


We showed 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 desired receptor and chromoprotein. We successfully demonstrated this concept for both PctA-Tar chimera and Histamine-Tar variant of the Rosetta software.

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).



To conclude, we showed a working concept of the FlashLab project, by using a commercial ibidi chip filled with a suspension of bacteria expressing the chimera and chromoprotein. A solution of a repellent/attractant was added to the chip and the displacement of the bacteria was monitored and recorded.




PctA-Tar chimera


We have successfully confirmed the functionality of the hybrid PctA-Tar receptor using swarming assay. From the results seen below, figure 3, 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. 3: 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.


PctA-Tar fused with GFP marker:

In addition, the correct localization of the chimera on both poles of the bacteria membrane, was proven through fusion of GFP to the C- terminus of the chimera with a short linker sequence (K1992010). The results of these tests as seen in figure 4, indeed show the expected localizations.


Fig. 4: 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 above, video 2, 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.

Trap and Track - Novel assay for digital chemotaxis detection

Although there is an abundant number of chemotaxis assays available today, most of them were designed 40 to 50 years ago and almost none provide a real time measurement without the use of fluorescence labeling.
The use of Porous Si (PSi) and oxidized PSi (PSiO2) matrices for biological sensing is on the rise. So far various analytes such as DNA, proteins and bacteria have been proven to be detectable on such matrices. The common method to monitor the interaction of said analytes within the porous films is reflective interferometric Fourier transform spectroscopy (RIFTS), as it allows a real time measurement and output for the user.
We present the results of an early experiment for the detection of chemotactic activity on the porous silicon films initially developed for bacterial detection.

This assay provides us with the means to digitally quantify chemotaxis - meaning we have a clear distinction between an attractant response (negative trend), repellent response (positive trend) and no response (zero trend).

The following graph presents only the chemotactic responses taken from 4 different experiments, The top 2 plots, in blue and in grey, represent a repellent response. The yellow plot represents a negative control experiment and the bottom red graph represents an attractant response.
For more information see our Measurement page.

Fig. 5:Digital chemotaxis measurements.

References:
1. 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




S.tar, by iGEM Technion 2016