Team:USTC-China/modeling/motion

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     <div class="title"><h1>Results</h1>
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      <h1>Motion</h1>
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      <dd data-magellan-arrival="lightsensingimagingsystem"><a herf="#lightsensingimagingsystem">Light Sensing-Imaging System</a></dd>
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            <dd data-magellan-arrival="introduction"><a href="#introduction">Introduction</a></dd>
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  <dd data-magellan-arrival="calobactercrescentus"><a herf="#calobactercrescentus">Caulobacter crescentus</a></dd>
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            <dd data-magellan-arrival="analysisoftheproblem"><a href="#analysisoftheproblem">Analysis</a></dd>
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<dd data-magellan-arrival="gettingthehelpfromeinstein"><a href="#gettingthehelpfromeinstein">Getting the help from Einstein</a></dd>
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<dd data-magellan-arrival="modelingresults"><a href="#modelingresults">Results</a></dd>
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    <a name="lightsensingimagingsystem"></a>
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<h3 data-magellan-destination="lightsensingimagingsystem">Light Sensing-Imaging System</h3>
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      <a name="introduction"></a>
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    <p><strong>Construction of Blue Light Sensing and Imaging System</strong></p>
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      <h3 data-magellan-destination="introduction">Introduction</h3>
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     <p>In our project, we want to stop the movement of C.crescentus to image a stable photo.  So we need to know whether we have stopped those excited boys.  One problem about this is, how to define and measure the parameter of  the C.crescentus' motion?</p>
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     <p>Because of the complication of blue light circuit, work on this circuit began firstly. In the beginning, K592020 which is a blue light sensor output containing a FixK2 blue light sensing system and producing amilCP was constructed.</p>
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      <a name="analysisoftheproblem"></a>
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    <p><img style="height: 300px;" src="/content/images/2014/Oct/QQ--20141006104132.jpg" alt="" /></p>
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    <h3 data-magellan-destination="analysisoftheproblem">Analysis</h3>
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     <p>Here we make the hypothesis below.</p>
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     <p>The culture of bacteria went like this: <br />
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    <ol>
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     <img src="/content/images/2014/Oct/K592020.jpg" alt="" /></p>
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    <li>the solution is uniform, which means the composition of every part in the solution is the same  </li>
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    <li>the number of the bacteria would not change in short time.  </li>
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    <li>the motion of the C.crescentus is random and they can be regarded as free gas.</li>
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    </ol>
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    <p>And we suppose  the distance one bacterium moved in time $t$ is $l$.  Then the diffusion coefficient D can be defined as <br />
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     $$D=l^2/(2t)$$
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        <img src="https://static.igem.org/mediawiki/2014/b/b0/USTC-China_Modeling_Motion_Diffusion.jpg" class="th"/></p>
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    <p>As you can guess, we are going to use $D$ to describe the motion of C.crescentus in stead of velocity. The reason is that the object of our study is a large crowd of bacteria, and it is more suitable to use diffusion coefficient as the parameter of the system.</p>
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     <p>In fact, $D$ describe the diffusion rate of bacteria. It's easy to predict that after we give the signal "stop", $D$ would decline because the C.crescentus can not get the help from the rotation of their flagellum and the holdfast would block their movement.</p>
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     <p>And this is the bands exhibition of K592020 and K592016, another blue light sensor and RR with RBS, after electrophoresis, quite satisfied as we expected. <br />
 +
    <img src="/content/images/2014/Oct/Marker-K592016-K592020-2.jpg" alt="" /></p>
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     <p>But, how can we measure the value of the diffusion coefficient?</p>
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     <p>The electrophoresis result of other key parts in blue light sensing-imaging system including B0015, S04617 and K592024. The part I13502 which expresses mRFP was run with them. <br />
 +
    <img src="/content/images/2014/Oct/Marker-B0015-S04617-I13502-K59202-2.jpg" alt="" /></p>
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    <a name="gettingthehelpfromeinstein"></a>
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     <p>Later, construction of the whole parts completed with validation of sequence verification.</p>
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<h3 data-magellan-destination="gettingthehelpfromeinstein">Getting the help from Einstein</h3>
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        <img src="https://static.igem.org/mediawiki/2014/5/54/IGEM_USTC-China_Modeling.jpg" class="th"/>
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        <img src="https://static.igem.org/mediawiki/2014/f/fa/USTC-China_Modeling_Motion_RW1.jpg" height="275px" class="th" />
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     <p>In early years of last century, Einstein focused on the motion of free gas, and got the famous Einstein Relation:
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    $$Ave(x^2)=2Dt$$
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    where $x$ is the distance one bacterium moved in time $t$, and $Ave(x^2)$ is the average value of x square. The relation shows that $Ave(x^2)$ is in direct proportion to the time $t$. </p>
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     <p>With this theory, we can develop a method to measure the value of $D$. Firstly we use micro camera to record the motion of bacteria. And then we focus on several bacteria's movement locus to count $Ave(x^2)$ with time $t$. Using these statistic data we can plot the relation between $Ave(x^2)$ and $2t$, and  the slope of the plot is what we want, the value of D.</p>
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     <p><strong>Test of Blue Light Sensing and Imaging System</strong></p>
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     <h3 id="modelingresults"></a>
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     <p>1.Test in solid media.</p>
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<h3 data-magellan-destination="modelingresults">Results</h3>
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     <img src="https://static.igem.org/mediawiki/2014/e/ea/Ustc-china-motion1.png" class="th"/>
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     <p>Without induced expression, bacteria containing blue light sensing-imaging system grew up and developed in the LB media. Then we divided the media into two regions, one of them receive blue light simulation while the other one keep darkness by a base plate to block blue light. The testing conditions showed in the following:</p>
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        <p>We use Mathematics to simulate 500-step movements of 5 bacteria, the image above shows their locus (in different color). ALl the bacteria "moved" randomly to different directions. Than we calculate square of the distance from original point, plot it with steps $ n $  <br />
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    <img src="https://static.igem.org/mediawiki/2014/a/a8/IGEM_USTC-China_Modeling_RandomWalk_Results.jpg" class="th" /></p>
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     <p>From the figure we can conclude that different diffusion coefficients correspond to different slopes of the fitting line. So we can develop a experimental method to evaluate the parameter of the system.</p>
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     <p><img src="/content/images/2014/Oct/DSCF1206.jpg" alt="" /></p>
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     <blockquote>
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     <p>After 12 hours development, the result showed the bacteria could produce blue chromoprotein when stimulated by blue light and keep its color in the darkness.</p>
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      <ul>
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      <li>This work is chiefly done by <strong>Hongda Jiang</strong>, with the assistance of <strong>Fangming Xie</strong>.</li>
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      <li>This article is written by <strong>Hongda Jiang</strong>, edited by <strong>Fangming Xie</strong>.</li>
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      </ul>
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    </div>
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    <p><img src="/content/images/2014/Oct/Sense-Result-2.jpg" alt="" /></p>
 +
 +
    <p>2.Test in liquid media.</p>
 +
 +
    <p>The idea of control-expriment is the same as in solid media. We prepared two test tubes containing equivalent bacterium solution. The test was conducted at 18:00, August 4th when one of them was put under blue light and the other was developed in the darkness.  </p>
 +
 +
    <p>At 11:00, August 5th, the tube stimulated under blue light turned light blue while the other one kept the original color.  </p>
 +
 +
    <p>After 24 hours, The blue bacteria showed evidently. Using centrifuge to get the bacteria showed extremely distinct blue chromoprotein expression base on blue light activation was come true. <br />
 +
    <img src="/content/images/2014/Oct/Comparasaion-2.jpg" alt="" /></p>
 +
 +
    <p><strong>Test of Green and Red Light Sensing-Imaging System</strong></p>
 +
 +
    <p>The procedure of experiment is the same as blue light test. The test started at 19:00, August 5th. After 22 hours growing up, bacteria after centrifuge appear red. but not obvious like blue and green. However, the GFP seemed not influence by light stimulation, which means it was also green in the darkness.</p>
 +
 +
    <p><img src="/content/images/2014/Oct/DSCF1212.jpg" alt="" />
 +
    <img src="/content/images/2014/Oct/Result.jpg" alt="" /></p>
 +
 +
    <p>The whole result shows in the following, and 2 days later the OD600nm of all tubes is in the following as well. <br />
 +
    <img src="/content/images/2014/Oct/OD-.png" alt="" /></p>
 +
 +
    <p><strong>Pattern Test</strong></p>
 +
 +
    <p>To test whether bacteria could produce a pattern with light stimulation, an experiment using projector to generate a pattern was conducted.</p>
 +
 +
    <p>The pattern was this: <br />
 +
    <img src="/content/images/2014/Oct/Testing-picture.jpg" alt="" /></p>
 +
 +
    <p>And the whole device like this: <br />
 +
    <img src="/content/images/2014/Oct/Working-Device.jpg" alt="" /></p>
 +
 +
    <p>Because blue light sensing-imaging system works well in our project, we used it to produced a shadow in the media and then added other bacteria later, just like engraving the picture in the earliest photographing. </p>
 +
 +
    <p><img src="/content/images/2014/Oct/Result2.jpg" alt="" /></p>
 +
 +
    <a name="calobactercrescentus"></a>
 +
<h3 data-magellan-destination="calobactercrescentus">Caulobacter crescentus</h3>
 +
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    <h4 id="conjugation">Conjugation</h4>
 +
 +
    <p><strong>Conjugation protocol</strong></p>
 +
 +
    <p><strong>conjugation of red light sensing-imaging system</strong></p>
 +
 +
    <p><strong>verification of <em>C.crescentus</em></strong></p>
 +
 +
    <p><strong>Growth Curve for Conjugation</strong></p>
 +
 +
    <p>To find the optimum of conjugation conditions, we test the development of S17-1 and <em>C.crescentus</em> in liquid PYE at 30 degree centigrade, which is almost the same condition while conjugation though the real conjugation is in solid PYE. We did so trying to get the result quantitively. <br />
 +
    <img src="/content/images/2014/Oct/QQ--20141008190727.png" alt="" /></p>
 +
 +
    <h4 id="motioncontrol">Motion Control</h4>
 +
 +
    <p>As we expected, DgrA, DgrB used for control the flagellum rotation and HfiA control the  synthesis of lipid which is key component of holdfast.</p>
 +
 +
    <p>The electrophoresis results like this.</p>
 +
 +
    <p><strong>DgrA and DgrB</strong></p>
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    <p><strong>HfiA</strong></p>
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Revision as of 04:52, 17 October 2014

Results

Light Sensing-Imaging System

Construction of Blue Light Sensing and Imaging System

Because of the complication of blue light circuit, work on this circuit began firstly. In the beginning, K592020 which is a blue light sensor output containing a FixK2 blue light sensing system and producing amilCP was constructed.

The culture of bacteria went like this:

And this is the bands exhibition of K592020 and K592016, another blue light sensor and RR with RBS, after electrophoresis, quite satisfied as we expected.

The electrophoresis result of other key parts in blue light sensing-imaging system including B0015, S04617 and K592024. The part I13502 which expresses mRFP was run with them.

Later, construction of the whole parts completed with validation of sequence verification.

Test of Blue Light Sensing and Imaging System

1.Test in solid media.

Without induced expression, bacteria containing blue light sensing-imaging system grew up and developed in the LB media. Then we divided the media into two regions, one of them receive blue light simulation while the other one keep darkness by a base plate to block blue light. The testing conditions showed in the following:

After 12 hours development, the result showed the bacteria could produce blue chromoprotein when stimulated by blue light and keep its color in the darkness.

2.Test in liquid media.

The idea of control-expriment is the same as in solid media. We prepared two test tubes containing equivalent bacterium solution. The test was conducted at 18:00, August 4th when one of them was put under blue light and the other was developed in the darkness.

At 11:00, August 5th, the tube stimulated under blue light turned light blue while the other one kept the original color.

After 24 hours, The blue bacteria showed evidently. Using centrifuge to get the bacteria showed extremely distinct blue chromoprotein expression base on blue light activation was come true.

Test of Green and Red Light Sensing-Imaging System

The procedure of experiment is the same as blue light test. The test started at 19:00, August 5th. After 22 hours growing up, bacteria after centrifuge appear red. but not obvious like blue and green. However, the GFP seemed not influence by light stimulation, which means it was also green in the darkness.

The whole result shows in the following, and 2 days later the OD600nm of all tubes is in the following as well.

Pattern Test

To test whether bacteria could produce a pattern with light stimulation, an experiment using projector to generate a pattern was conducted.

The pattern was this:

And the whole device like this:

Because blue light sensing-imaging system works well in our project, we used it to produced a shadow in the media and then added other bacteria later, just like engraving the picture in the earliest photographing.

Caulobacter crescentus

Conjugation

Conjugation protocol

conjugation of red light sensing-imaging system

verification of C.crescentus

Growth Curve for Conjugation

To find the optimum of conjugation conditions, we test the development of S17-1 and C.crescentus in liquid PYE at 30 degree centigrade, which is almost the same condition while conjugation though the real conjugation is in solid PYE. We did so trying to get the result quantitively.

Motion Control

As we expected, DgrA, DgrB used for control the flagellum rotation and HfiA control the synthesis of lipid which is key component of holdfast.

The electrophoresis results like this.

DgrA and DgrB

HfiA

Lots of thanks to our sponsors, who help us to achieve what we have today

© 2014 USTC-iGEM Design. Distributed under BY-SA 3.0

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