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

 
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<img src="https://static.igem.org/mediawiki/2016/d/db/T--Technion_Israel--icon_intro.png" class="img-responsive img-center cont_tabs" width="100" height="100">
 
<img src="https://static.igem.org/mediawiki/2016/d/db/T--Technion_Israel--icon_intro.png" class="img-responsive img-center cont_tabs" width="100" height="100">
<br><h4 class="text-center"><b>Introduction</b></h4>
+
<br><h4 class="text-center">Introduction</h4>
 
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<img src="https://static.igem.org/mediawiki/2016/4/49/T--Technion_Israel--icon_lab.png" class="img-responsive img-center cont_tabs" width="100" height="100">
<br><h4 class="text-center"><b>Implementation</b></h4>
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<br><h4 class="text-center">Implementation</h4>
 
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<img src="https://static.igem.org/mediawiki/2016/4/45/T--Technion_Israel--icon_results.png" class="img-responsive img-center cont_tabs" width="100" height="100">
<br><h4 class="text-center"><b>Results</b></h4>
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<br><h4 class="text-center">Results</h4>
 
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<img src="https://static.igem.org/mediawiki/2016/4/47/T--Technion_Israel--icon_outlook.png" class="img-responsive img-center cont_tabs" width="100" height="100">
<br><h4 class="text-center"><b>Outlook</b></h4>
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<br><h4 class="text-center">Outlook</h4>
 
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<p class="text-justify">
 
<p class="text-justify">
 
Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope.  
 
Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope.  
However, the FlashLab technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance, a cluster is formed and the presence of chromogenic proteins allows the user to spot it in the naked eye without the need for a complex device (for more information about our chip click <a href="https://2016.igem.org/Team:Technion_Israel/Design" target="_blank">here</a>).
+
However, the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance - a cluster is formed, and the presence of chromogenic proteins allows the user to spot it in the naked eye, without the need for a complex device (for more information about our chip click <a href="https://2016.igem.org/Team:Technion_Israel/Design" target="_blank">here</a>).
 
</p>
 
</p>
 
</div>
 
</div>
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<div class="col-md-12 col-sm-12">
 
<div class="col-md-12 col-sm-12">
 
<p class="text-justify">
 
<p class="text-justify">
Three chromogenic proteins were tested for the S.Tar system, all which were provided and extracted from the iGEM 2016 kit. <br>
+
Three chromogenic proteins (chromoproteins) were tested for the <a href="https://2016.igem.org/Team:Technion_Israel/S.Tar_intro">S.Tar</a>
These proteins were: <br>
+
system, all which were provided and extracted from the iGEM 2016 kit.  
1. K1357008 expressing purple color.<br>
+
Each part contained RBS, chromoproteins coding sequence and a double
2. K1357009 expressing blue color.<br>
+
terminator.  The different parts contained the following proteins:<br>
3. K1357010 expressing red color with fluorescent capabilities<br><br>
+
- tsPurple, visible as purple color <a href="http://parts.igem.org/Part:BBa_K1357008" target="_blank">(K1357008)</a>.<br>
 +
- amilCP, visible as blue color (<a href="http://parts.igem.org/Part:BBa_K1357009" target="_blank">K1357009</a>).<br>
 +
- mRFP, visible as red color and can serve, also ,as red fluorescence protein (<a href="http://parts.igem.org/Part:BBa_K1357010" target="_blank">K1357010</a>).<br>
 +
<br>
 +
To test the expression and visibility of those proteins, a strong promoter
 +
(<a href="http://parts.igem.org/Part:BBa_J23100" target="_blank">J23100</a>) was cloned
 +
upstream to the RBS using the RFC10 assembly (Fig. 1).
  
To test these proteins, a strong promoter (J23100) was added, with the help of restriction enzymes to each of them. In parallel, two of these proteins were added downstream of the Tar on the plasmid to test the expression level of both under the same promoter (figure 1).
 
 
 
 
 
</p>
 
</p>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="row">
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<div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
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<a class="pop ocenter">
 +
<img src="https://static.igem.org/mediawiki/2016/thumb/e/e8/T--Technion_Israel--chromoproteincircute.png/800px-T--Technion_Israel--chromoproteincircute.png" class="img-responsive img-center" width="450" style="cursor: pointer;">
 +
</a>
 +
<p class="text-center"><b>Fig. 1:</b>  High expression system of chromogenic protein.
 +
</p>
 +
</div>
 +
</div>
 +
 +
<!-- 12 text div -->
 +
<div class="row">
 +
<div class="col-md-12 col-sm-12">
 +
<p class="text-justify">
 +
This plasmid is one of two plasmids constructing our <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> system, as
 +
the other is plasmid expressing a chemoreceptor. The two plasmids were
 +
co-transformed to UU1250 strain expressing both, the designed
 +
receptor and a chosen color. <br>Each plasmid contains different antibiotic
 +
resistance allowing easy screening for strain expressing both proteins.
 +
</p>
 +
</div>
 +
</div>
 +
 +
  
  
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<p class="text-justify">
 
At the beginning we looked for chromogenic proteins in the iGEM Kit 2016. We found a list
 
of chromogenic protein and start to work with two chromogenic protein, K1357008 ( purple) 
 
& K1357009 (blue) , and one protein that have both, chromogenic and fluorescent abilities,
 
K1357010. We decided to test three different color hoping that one of them will suit our system.<br>
 
</p>
 
</div>
 
</div>
 
 
<br>
 
  
 
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<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
 
<p class="text-justify">
 
<p class="text-justify">
The first step was adding a strong promoter, J23100, to each color plasmid. For that we used
+
The first step, as mentioned in the implementation section,  
restriction enzymes (see: <a href="#" target="_blank">notebook</a>).
+
was to clone a strong promoter (<a href="http://parts.igem.org/Part:BBa_J23100" target="_blank">J23100</a>) upstream to each part,
In parallel we designed two plasmids that contain Tar and one of the chromogenic protein
+
creating a high expression system. The biological system was
under the same promoter.
+
then transformed to <i>E.coli</i> Top10 strain and UU1250 strain.
 +
Plating results showed colored colonies, for both strains,
 +
as expected. Colored colony from each type was incubated
 +
overnight at 37&#8451; in LB medium. Overnight incubation resulted in a
 +
medium that appeared as colored, due to high concentration of  
 +
bacteria expressing chromoproteins. The results of centrifuging the medium
 +
sample was a colored pellet (Fig. 1).
 +
 
 
</p>
 
</p>
 
</div><!--
 
</div><!--
 
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
 
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
 
<a class="pop ocenter">
 
<a class="pop ocenter">
<img src="#" class="img-responsive img-center" style="cursor: pointer;">
+
<img src="https://static.igem.org/mediawiki/2016/c/c6/T--Technion_Israel--color2.png" class="img-responsive img-center img-cont" width="450" style="cursor: pointer;">
 
</a>
 
</a>
<p class="text-center"><b>Fig. 1:</b> schematic color plasmide.
+
<p class="text-center"><b>Fig. 1:</b> <i>E.coli</i> Top10 strain expressing (left to right): mRFP - visible as red color, tsPurple - visible as purple color, amilCP - visible as blue color.</p>
</p>
+
 
</div>
 
</div>
</div>
+
</div>
 
+
<br>
<br>
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<br>
<br>
+
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<br>
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<div class="row">
 
<div class="row">
<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
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<div class="col-md-12 col-sm-12">
 
<p class="text-justify">
 
<p class="text-justify">
After transformation we could see colored bacteria on agar plats but when we tried to
+
As both strains showed similar results, the following experiments conducted
make an overnight culture for the next step we could not see the color in the Tar+color
+
only with the UU1250 strain, the strain which was used for chemotaxis assays.
strians. We centrifuge the culture and discovered that we have colored bacteria but they
+
Growth conditions for chemotaxis assays require a minimal growth medium,
not dense enough for us to see in the naked eye (fig 2).
+
TB, and a temperature of 30&#8451;. Above this temperature the bacteria lose their chemotaxis ability.(1).<br> Overnight growing in this condition,
</p>
+
of UU1250 strain expressing chromoproteins resulted in a colorless medium,
</div><!--
+
although bacterial concentration was high. In order to overcome this issue, two different growth conditions were tested. Incubation
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
+
at 37&#8451; in TB medium and incubation at 30&#8451; in LB medium.
<a class="pop ocenter">
+
At 37&#8451; TB medium, color was detected. The color was less intense
<img src="https://static.igem.org/mediawiki/2016/c/c6/T--Technion_Israel--color2.png" class="img-responsive img-center img-cont" style="cursor: pointer;" width="250">
+
compared to the 37&#8451; LB medium, but still high enough to be detected by
</a>
+
a naked eye. Moreover, the pellet's color intensity was similar to the one grown 37&#8451;
<p class="text-center"><b>Fig. 2:</b> <I>E.coli</I> Top 10 with (from right) K1357010 (RFP),  
+
LB pellet. As for the 30&#8451; LB medium, no color was detected after
K1357008 (purple) and K1357009 (blue) after centrifuge.
+
overnight growth. In addition, the pellet was also colorless.
 +
<br>These results imply that the growth temperature
 +
has a significant influence on the chromoprotein expression.
 +
To achieve color intensity at the right conditions a two-stage
 +
growth was conducted. The first stage is incubation at 37&#8451;  
 +
in LB medium in order to gain a high expression of chromoproteins.
 +
The culture is then centrifuged and resuspend with TB medium.
 +
The second stage is incubation at 30&#8451; for 3 hours, for restoring
 +
chemotaxis abilities. This two-stage growth allows both color
 +
expression and chemotaxis ability to the bacteria, and
 +
was proven to be effective in matter for chromoprotein
 +
expression, as for chemotaxis ability test, the two palsmid system was conducted.
 
</p>
 
</p>
 
</div>
 
</div>
</div>
+
</div>
 
+
<br>
+
<br>
+
<br>
+
  
 +
<!-- Mini headline -->
 
<div class="row">
 
<div class="row">
 
<div class="col-md-12 col-sm-12">
 
<div class="col-md-12 col-sm-12">
<p class="text-justify">
+
<h3>The two plasmid system</h3>
We didn't give up on the idea. Instead we tried to play we the condition in which
+
our overnight culture is growing. Our basic chemotaxis experiment, swarming assay,
+
require a minimal growth medium, TB, and an optimal temperature of 30 degree.
+
Optimal growth of <I> E.coli</I> is 37 degree <b>(makor safrot kan on the Tem.)</b>. So we played
+
in that range hoping to discover an optimal condition for both, chemotaxis and
+
color formation.
+
</p>
+
 
</div>
 
</div>
</div>
+
</div>
 
+
<br>
<br>
+
 
+
 
<!--6 text - 6 img div-->
 
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<div class="row">
 
<div class="row">
 
<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
 
<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
 
<p class="text-justify">
 
<p class="text-justify">
It was easy to get and see the color in 37C (fig 3) with LB medium, just by letting
+
<a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> system is based on a two-plasmid system, where motile  bacteria express both
the culture enough time. For the same temperature and time in TB we couldn’t see the
+
chromoproteins, using two different expression plasmids. In order to verify the idea, Tar was chosen as the chemoreceptor and amilCP (blue color) as the chromoprotein. The first
color. Initially we thought that the TB medium is not rich enough for colored protein
+
plasmid (<a href="http://parts.igem.org/Part:BBa_K1992004" target="_blank">K1992004</a>) expresses the Tar chemoreceptor,  
formation. We check the O.D and saw a significant difference between the LB and the
+
along with chloramphenicol (CM) resistance. The second
TB medium. After almost 24 hours of incubation we could finally see the color in the  
+
plasmid expresses a chromoprotein (see Implementation –
TB tubs.
+
part have not been submitted) along with ampicillin (Amp)
 +
resistance. Follolwing transformation of the two plasmids into UU1250 strain (which lacks chemoreceptors genes), the colonies were selected by plating on LB agar plates with two antibiotic. The plating results
 +
showed colored colonies and non-colored ones (Fig. 2), that is due to the  
 +
non-compatible ORI of the two plasmids (see Outlook section).  
 
</p>
 
</p>
 
</div><!--
 
</div><!--
 
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
 
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
 
<a class="pop ocenter">
 
<a class="pop ocenter">
<img src="https://static.igem.org/mediawiki/2016/8/8e/T--Technion_Israel--color3.png" class="img-responsive img-center img-cont" style="cursor: pointer;" width="250">
+
<img src="https://static.igem.org/mediawiki/2016/a/ac/T--Technion_Israel--colornew1.jpg" class="img-responsive img-center img-cont" width="450" style="cursor: pointer;">
 
</a>
 
</a>
<p class="text-center"><b>Fig. 3:</b> overnight starters 37C, in LB. In the tubs:  
+
<p class="text-center"><b>Fig. 2:</b> Co-transformation of <a href="http://parts.igem.org/Part:BBa_K1992004" target="_blank">K1992004</a> and tsPurple expressing plasmid to UU1250 strain.</p>
Top 10 RFP, UU1250 with the purple plasmid and UU1250 with blue plasmid.
+
</p>
+
 
</div>
 
</div>
</div>
+
</div>
 
+
<br>
<br>
+
<!-- 12 text div -->
<br>
+
<br>
+
 
+
<!--6 text - 6 img div-->
+
 
<div class="row">
 
<div class="row">
<div class="col-md-6 col-sm-12 vcenter"><!--6 text-->
+
<div class="col-md-12 col-sm-12">
 
<p class="text-justify">
 
<p class="text-justify">
Next we played with the temperature. As mention above the optimal temperature for
+
The colored colonies were isolated and grown, using the two-stage
chemotaxis is 30 degree in this temperature bacteria growth is slow and color
+
growth method mentioned previously. The result is high density
formation even slower. We tried to grow the bacteria in 37 degree and then move
+
and colored medium (Fig. 3).
it to 30 degree for few generation. Due we could see chemotaxis after that process
+
the bacteria didn't always make enough protein for as to see in logical time period.
+
That will affect our final product so we have to find another solution (table 1).
+
 
</p>
 
</p>
</div><!--
+
</div>
--><div class="col-md-6 col-sm-12 vcenter"><!--6 img div-->
+
</div>
 +
<br>
 +
<!-- 12 img div -->
 +
<div class="row">
 +
<div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
 
<a class="pop ocenter">
 
<a class="pop ocenter">
<img src="https://static.igem.org/mediawiki/2016/e/ee/T--Technion_Israel--color4.PNG" class="img-responsive img-center img-cont" style="cursor: pointer;" width="450">
+
<img src="https://static.igem.org/mediawiki/2016/0/08/T--Technion_Israel--colornew3.jpeg" class="img-responsive img-center img-cont" width="250" style="cursor: pointer;">
 
</a>
 
</a>
<p class="text-center"><b>Table 1:</b> color formation test results. N.D - no data, N.C - no color.
+
<p class="text-center"><b>Fig. 3:</b> Left tube - UU1250 strain expressing Tar
 +
chemoreceptor only (<a href="http://parts.igem.org/Part:BBa_K1992004" target="_blank">K1992004</a>). Right tube - UU1250 strain expressing Tar
 +
chemoreceptor and tsPurple chromoprotein.
 
</p>
 
</p>
 
</div>
 
</div>
</div>
+
</div>
 
+
<br>
<br>
+
<!-- 12 text div -->
<br>
+
<br>
+
 
+
 
<div class="row">
 
<div class="row">
 
<div class="col-md-12 col-sm-12">
 
<div class="col-md-12 col-sm-12">
 
<p class="text-justify">
 
<p class="text-justify">
We didn’t want to play with the order of the parts as we were afraid we will
+
The sample was then tested for chemotaxis ability using
harm the chemotaxise ability of the bacteria so we decided to separate and work
+
<a href="https://2016.igem.org/Team:Technion_Israel/Experiments">swarming assay</a>, in which the chemotaxis ability is examined in a swarm plate. The bacteria depleting the attractant and move outwards, creating a halo. (Fig. 4). A halo was formed after
with two plasmids, one with the color protein and second with our receptor. In
+
8 hours indicating functional chemotaxis response. For our
this approach we got the desirable color in the overnight culture whiteout farther treatment.    
+
<a href="https://2016.igem.org/Team:Technion_Israel/Experiments">chip assay</a> more intense color was needed. Thus, the sample was centrifuged and resuspended in a smaller volume
 +
of TB medium, increasing the bacterial concentration by 10-folds.
 +
Results can be seen in the <a href="https://2016.igem.org/Team:Technion_Israel/Proof">Proof of concept page</a>.
 
</p>
 
</p>
 
</div>
 
</div>
</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/2016/9/99/T--Technion_Israel--colornew2.png" class="img-responsive img-center img-cont" width="700" style="cursor: pointer;">
 +
</a>
 +
<p class="text-center"><b>Fig. 4:</b>
 +
Swarming assay results (left to right) - UU1250 expressing only Tar chemoreceptor;
 +
UU1250 expressing Tar chemoreceptor and tsPurple chromoprotein;
 +
UU1250 only; &Delta;Zras - <i>E.coli</i> expressing all chemoreceptors.
 +
</p>
 +
</div>
 +
</div>
 +
 +
 +
  
 
</div>
 
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<div class="col-md-12 col-sm-12">
 
<div class="col-md-12 col-sm-12">
 
<p class="text-justify">
 
<p class="text-justify">
We succeed to get colored bacteria that are comfortable to work with in the optimal  
+
We succeeded to get colored bacteria, grown in the optimal condition of our assay,  
condition to our assay. At that point both, the color and the receptors, cloned on  
+
though both the chemoproteins and the receptors were cloned on high copy
high copied plasmid. Every time the culture grow the bacteria copied one of the  
+
plasmid with the same ORI - pMB1 (pSB1C3 and pSB1A2). Usually, plasmids with the  
plasmid as a high copy and the second one as low copy. By moving the receptor to  
+
same ORIs are incompatible, because they will compete for the same machinery,
low copy plasmid we could achieve constant color bacteria and enough receptors
+
creating an unstable and unpredictable environment. For future plan, we suggest to  
for bacterial movement.
+
clone one of the expression systems to a plasmid containing different ORI,
 +
compatible to pMB1 ORI. This adjustment will improve the stability of our
 +
system, and allow better control over the expression of each protein.
 
</p>
 
</p>
 
</div>
 
</div>

Latest revision as of 03:57, 20 October 2016

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 2016

Introduction


Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope. However, the FlashLab technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance - a cluster is formed, and the presence of chromogenic proteins allows the user to spot it in the naked eye, without the need for a complex device (for more information about our chip click here).

Implementation


Three chromogenic proteins (chromoproteins) were tested for the S.Tar system, all which were provided and extracted from the iGEM 2016 kit. Each part contained RBS, chromoproteins coding sequence and a double terminator. The different parts contained the following proteins:
- tsPurple, visible as purple color (K1357008).
- amilCP, visible as blue color (K1357009).
- mRFP, visible as red color and can serve, also ,as red fluorescence protein (K1357010).

To test the expression and visibility of those proteins, a strong promoter (J23100) was cloned upstream to the RBS using the RFC10 assembly (Fig. 1).

Fig. 1: High expression system of chromogenic protein.

This plasmid is one of two plasmids constructing our FlashLab system, as the other is plasmid expressing a chemoreceptor. The two plasmids were co-transformed to UU1250 strain expressing both, the designed receptor and a chosen color.
Each plasmid contains different antibiotic resistance allowing easy screening for strain expressing both proteins.

Results


The first step, as mentioned in the implementation section, was to clone a strong promoter (J23100) upstream to each part, creating a high expression system. The biological system was then transformed to E.coli Top10 strain and UU1250 strain. Plating results showed colored colonies, for both strains, as expected. Colored colony from each type was incubated overnight at 37℃ in LB medium. Overnight incubation resulted in a medium that appeared as colored, due to high concentration of bacteria expressing chromoproteins. The results of centrifuging the medium sample was a colored pellet (Fig. 1).

Fig. 1: E.coli Top10 strain expressing (left to right): mRFP - visible as red color, tsPurple - visible as purple color, amilCP - visible as blue color.



As both strains showed similar results, the following experiments conducted only with the UU1250 strain, the strain which was used for chemotaxis assays. Growth conditions for chemotaxis assays require a minimal growth medium, TB, and a temperature of 30℃. Above this temperature the bacteria lose their chemotaxis ability.(1).
Overnight growing in this condition, of UU1250 strain expressing chromoproteins resulted in a colorless medium, although bacterial concentration was high. In order to overcome this issue, two different growth conditions were tested. Incubation at 37℃ in TB medium and incubation at 30℃ in LB medium. At 37℃ TB medium, color was detected. The color was less intense compared to the 37℃ LB medium, but still high enough to be detected by a naked eye. Moreover, the pellet's color intensity was similar to the one grown 37℃ LB pellet. As for the 30℃ LB medium, no color was detected after overnight growth. In addition, the pellet was also colorless.
These results imply that the growth temperature has a significant influence on the chromoprotein expression. To achieve color intensity at the right conditions a two-stage growth was conducted. The first stage is incubation at 37℃ in LB medium in order to gain a high expression of chromoproteins. The culture is then centrifuged and resuspend with TB medium. The second stage is incubation at 30℃ for 3 hours, for restoring chemotaxis abilities. This two-stage growth allows both color expression and chemotaxis ability to the bacteria, and was proven to be effective in matter for chromoprotein expression, as for chemotaxis ability test, the two palsmid system was conducted.

The two plasmid system


FlashLab system is based on a two-plasmid system, where motile bacteria express both chromoproteins, using two different expression plasmids. In order to verify the idea, Tar was chosen as the chemoreceptor and amilCP (blue color) as the chromoprotein. The first plasmid (K1992004) expresses the Tar chemoreceptor, along with chloramphenicol (CM) resistance. The second plasmid expresses a chromoprotein (see Implementation – part have not been submitted) along with ampicillin (Amp) resistance. Follolwing transformation of the two plasmids into UU1250 strain (which lacks chemoreceptors genes), the colonies were selected by plating on LB agar plates with two antibiotic. The plating results showed colored colonies and non-colored ones (Fig. 2), that is due to the non-compatible ORI of the two plasmids (see Outlook section).

Fig. 2: Co-transformation of K1992004 and tsPurple expressing plasmid to UU1250 strain.


The colored colonies were isolated and grown, using the two-stage growth method mentioned previously. The result is high density and colored medium (Fig. 3).


Fig. 3: Left tube - UU1250 strain expressing Tar chemoreceptor only (K1992004). Right tube - UU1250 strain expressing Tar chemoreceptor and tsPurple chromoprotein.


The sample was then tested for chemotaxis ability using swarming assay, in which the chemotaxis ability is examined in a swarm plate. The bacteria depleting the attractant and move outwards, creating a halo. (Fig. 4). A halo was formed after 8 hours indicating functional chemotaxis response. For our chip assay more intense color was needed. Thus, the sample was centrifuged and resuspended in a smaller volume of TB medium, increasing the bacterial concentration by 10-folds. Results can be seen in the Proof of concept page.

Fig. 4: Swarming assay results (left to right) - UU1250 expressing only Tar chemoreceptor; UU1250 expressing Tar chemoreceptor and tsPurple chromoprotein; UU1250 only; ΔZras - E.coli expressing all chemoreceptors.

Outlook


We succeeded to get colored bacteria, grown in the optimal condition of our assay, though both the chemoproteins and the receptors were cloned on high copy plasmid with the same ORI - pMB1 (pSB1C3 and pSB1A2). Usually, plasmids with the same ORIs are incompatible, because they will compete for the same machinery, creating an unstable and unpredictable environment. For future plan, we suggest to clone one of the expression systems to a plasmid containing different ORI, compatible to pMB1 ORI. This adjustment will improve the stability of our system, and allow better control over the expression of each protein.

References:
1. MAEDA, Kayo, et al. Effect of temperature on motility and chemotaxis of Escherichia coli. Journal of bacteriology, 1976, 127.3: 1039-1046.‏




S.tar, by iGEM Technion 2016