IGEM:Kyoto/2008/project: Difference between revisions

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<!--main-->
<!--main-->
  <p class="title">Cells as physical power suppliers:<p>
   <p class="title">Raise the Titanic!</p>
   <p class="title">Raise the Titanic!</p>


<!--Project Description-->
<!--Project Description-->
   <div class="project">
   <div class="project">
   <p class="subtitle">Project Description</p>
   <p class="subtitle">Project Description</p>
  <p class="main">In many biotechnological contexts, bacterial cells are considered as "chemical facilities." A number of studies have genetically engineered cells to produce various desired compounds. They further aim at accurate and precise regulation of material production. Cells are also power suppliers in terms of their motility. This aspect, however, has been much less featured. Here comes our project, which started with the gigantic goals of lifting up the Titanic from the deep-sea with bacterial power. Toward our general goal – to engineer cells to carry larger order of objects – we have been designing and constructing cells so that these micro-order entities can move a centimeter or larger objects. We have equipped E. coli with the functions of attachment to an object surface, cell density dependent buoyancy production, and regulatable flagella and examined by quantitating the parameters to what extent our goal is achieved. Our study presents the possibility of bacterial physical power.</p>
  </div>
<!--YouTube-->
  <div align="center" class="youtube">
  <object width="425" height="349"><param name="movie" value="http://www.youtube.com/v/inU2rg6QyGA&hl=ja&fs=1&rel=0&color1=0x3a3a3a&color2=0x999999&border=1"></param><param name="allowFullScreen" value="true"></param><embed src="http://www.youtube.com/v/inU2rg6QyGA&hl=ja&fs=1&rel=0&color1=0x3a3a3a&color2=0x999999&border=1" type="application/x-shockwave-flash" allowfullscreen="true" width="425" height="349"></embed></object>


  <p class="main">In many biotechnological contexts, bacterial cells are considered as "chemical facilities." A number of studies have genetically engineered cells to produce various desired compounds. They further aim at accurate and precise regulation of material production. Cells are also power suppliers in terms of their motility. This aspect, however, has been much less featured. Here comes our project, which started with the gigantic goals of lifting up the Titanic from the deep-sea with bacterial power. Toward our general goal – to engineer cells to carry larger order of objects – we have been designing and constructing cells so that these micro-order entities can move a centimeter or larger objects. We have equipped E. coli with the functions of attachment to an object surface, cell density dependent buoyancy production, and regulatable flagella and examined by quantitating the parameters to what extent our goal is achieved. Our study presents the possibility of bacterial physical power.</p>
   </div>
   </div>


<!--Systems-->
   <div class="project">
   <div class="project">
   <p class="subtitle">Function A: Binding to Ti and polystyrene<br>
   <p class="subtitle">Functions: <span style="font-family:serif;">Bind, Buoy, and Push!</span></p>
   (Modified genes: luxI, luxR, gvpA, gvpB, gvpC)</p>
 
  <p class="main">Our E. coli machine has these 3 functions.</p>
 
</div>
 
<!--functionA-->
  <div class="project">
 
  <li class="functions">Function A: Binding to Ti and polystyrene</li>
   <p class="genes">(Modified genes: luxI, luxR, gvpA, gvpB, gvpC)</p>
  <p class="functions">Fisrt, cells must bind to the surface of the Titanic. For this purpose, we employed the cell surface display method with Lpp-OmpA-fusion protein. This method enables to display particular peptides on the surface of the gram negative bacteria. In this project, we fused two kinds of binding peptides with OmpA; one is Titanium-binding peptide (TBP)3 and the other is polystyrene binding peptide (PBP)4. Both of which were obtained by PCR cloning.</p>


  <p class="main">To make cells stiffly bind to the surface of the Titanic, we use a famous method of cell surface display; Lpp-OmpA-fusion protein used display1,2. We can apply this method to display particular peptide on the surface of the gram negative bacteria.
In this case, we fused two affinity holding peptides; one is Titan binding peptide (TBP)3 and the other is polystyrene binding peptide (PBP)4. We cited the dominant sequence from following papers and applied it into PCR cloning.</p>
   </div>
   </div>


  <table class="image" align="center">
    <tr><td>
  <a href="http://openwetware.org/images/7/74/Kyoto-Binding.jpg" title="Image:Binding.jpg" target="_blank"><img alt="Binding" src="http://openwetware.org/images/7/74/Kyoto-Binding.jpg" border="0" width="300" height="250" /></a>
    </td></tr>
  </table>
<!--functionB-->
   <div class="project">
   <div class="project">
  <p class="subtitle">Function B: Cell density-dependent buoyancy<br>
  (Modified genes: luxI, luxR, gvpA, gvpB, gvpC)</p>


   <p class="main">E. coli is given buoyancy by interacellular hollow organelles called gas vesicle. The expression of gas vesicle is regulated by increased cell population. This system uses Quorum sensing that is cell to cell communication system. We transferred two genes luxI and luxR from V. fischeri to E.coli (iGEM parts). LuxI is an autoinducer synthase, which produces the acyl-homoserin lactone(AHL). LuxR is protein that can bind AHL. The AHL diffuse out of the cell and increases its concentration with increasing cell population. When bound to AHL, lux promoter stimulates and Gas vesicle appears.</p>
  <li class="functions">Function B: Cell density-dependent buoyancy</li>
   <p class="genes">(Modified genes: luxI, luxR, gvpA, gvpB, gvpC)</p>
  <p class="functions">We designed the cell density dependent buoyancy system by which cells produce buoyancy only when they have sufficiently increased. This aims at both fast growth rate and enough buoyancy. E. coli gains buoyancy from intracellular hollow organelles called gas vesicles. The expression of this gene is regulated by quorum sensing (QS) mechanism. Not activating gas vesicle genes during, for eample, an exponential growth phase enhances their grow rate. For QS, E. coli was transformed with two genes luxI and luxR . LuxI is an autoinducer synthase, which produces acyl-homoserin lactone (AHL). LuxR is a protein that binds to AHL. Since AHL freely diffuses though cell membrane, its concentration reflects density of the cells. When bound to AHL, LuxR stimulates Lux promoter of the gas vesicle gene. For gas vesicle proteins, only gvpA – C among at least 15 related genes are used to minimize the cost of the protein production.</p>
 
   </div>
   </div>


  <table class="image" align="center">
    <tr><td>
    <a href="http://openwetware.org/images/8/89/Kyoto-Quorumgas.jpg" target="_blank"><img alt="Quorumgas" src="http://openwetware.org/images/8/89/Kyoto-Quorumgas.jpg" border="0" width="300" height="250" /></a>
    </td></tr>
  </table>
<!--functionC-->
   <div class="project">
   <div class="project">
  <p class="subtitle">Function C: Light/dark-dependent flagella<br>
  Our engineered E. coli is designed more likely to continue counter clockwise (CCW) flagella rotatation in the darkness due to cheZ.</p>


   <p class="main">The mechanism consists of following four points:<br>
  <li class="functions">Function C: Light-dependent flagella rotation</li>
   <p class="genes">Our engineered E. coli is designed more likely to continue counter clockwise (CCW) flagella rotatation in the darkness due to cheZ.</p>
  <p class="functions">The mechanism consists of following four points:</p>
 
  <ol class="functionC">
    <li class="functionC">CCW is promoted in the presence of CheZ.</li>
    <li class="functionC">In our E.coli, the synthesis of CheZ is accelerated in the darkness and is repressed in the light.</li>
    <li class="functionC">Then, the number of E.coli rotating flagella CCW on the darker side of the object on which E.coli is binding exceeds that on the lighter side.</li>
    <li class="functionC">The object on which E.coli is binding can gain force in the direction of lighter place.</li>
  </ol>
 
  <p class="functions">alternative below</p>
 
  <p class="functions">Our E. coli is designed to sense light and then generate thrust as following.
 
  <ul class="functionC">
    <li class="functionC">CCW rotation of flagella produces thrust</li>
    <li class="functionC">CCW is promoted in the presence of CheZ.</li>
    <li class="functionC">In our E.coli, cheZ expression is activated by light.</li>
  </ul>
 
  <p class="functions">For the production of light activated protein. three genes, ho1, pcyA, and cph8 of BioBrick parts were introduced. This protein is phosphorylated in light exposure and activates a specific promoter and expression of its downstream gene cheZ.</p>


　1) CCW is promoted in the presence of CheZ.<br>
　2) In our E.coli, the synthesis of CheZ is accelerated in the darkness and is repressed in the light.<br>
　3) Then, the number of E.coli rotating flagella CCW on the darker side of the object on which E.coli is binding exceeds that on the lighter side.<br>
　4) The object on which E.coli is binding can gain force in the direction of lighter place.</p>
   </div>
   </div>
  <table class="image" align="center">
  <tr><td>
    <a href="http://openwetware.org/images/e/e2/Kyoto-Flagella.jpg" target="_blank"><img alt="Flagella" src="http://openwetware.org/images/e/e2/Kyoto-Flagella.jpg" border="0" width="300" height="250" /></a>
  </td></tr>
  </table>


  </div>
  </div>
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Latest revision as of 22:51, 13 November 2008

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Raise the Titanic!

Project Description

In many biotechnological contexts, bacterial cells are considered as "chemical facilities." A number of studies have genetically engineered cells to produce various desired compounds. They further aim at accurate and precise regulation of material production. Cells are also power suppliers in terms of their motility. This aspect, however, has been much less featured. Here comes our project, which started with the gigantic goals of lifting up the Titanic from the deep-sea with bacterial power. Toward our general goal – to engineer cells to carry larger order of objects – we have been designing and constructing cells so that these micro-order entities can move a centimeter or larger objects. We have equipped E. coli with the functions of attachment to an object surface, cell density dependent buoyancy production, and regulatable flagella and examined by quantitating the parameters to what extent our goal is achieved. Our study presents the possibility of bacterial physical power.

  <object width="425" height="349"><param name="movie" value="http://www.youtube.com/v/inU2rg6QyGA&hl=ja&fs=1&rel=0&color1=0x3a3a3a&color2=0x999999&border=1"></param><param name="allowFullScreen" value="true"></param><embed src="http://www.youtube.com/v/inU2rg6QyGA&hl=ja&fs=1&rel=0&color1=0x3a3a3a&color2=0x999999&border=1" type="application/x-shockwave-flash" allowfullscreen="true" width="425" height="349"></embed></object>

Functions: Bind, Buoy, and Push!

Our E. coli machine has these 3 functions.

  • Function A: Binding to Ti and polystyrene

    • (Modified genes: luxI, luxR, gvpA, gvpB, gvpC)

    Fisrt, cells must bind to the surface of the Titanic. For this purpose, we employed the cell surface display method with Lpp-OmpA-fusion protein. This method enables to display particular peptides on the surface of the gram negative bacteria. In this project, we fused two kinds of binding peptides with OmpA; one is Titanium-binding peptide (TBP)3 and the other is polystyrene binding peptide (PBP)4. Both of which were obtained by PCR cloning.

      <a href="http://openwetware.org/images/7/74/Kyoto-Binding.jpg" title="Image:Binding.jpg" target="_blank"><img alt="Binding" src="http://openwetware.org/images/7/74/Kyoto-Binding.jpg" border="0" width="300" height="250" /></a>
  • Function B: Cell density-dependent buoyancy

    • (Modified genes: luxI, luxR, gvpA, gvpB, gvpC)

    We designed the cell density dependent buoyancy system by which cells produce buoyancy only when they have sufficiently increased. This aims at both fast growth rate and enough buoyancy. E. coli gains buoyancy from intracellular hollow organelles called gas vesicles. The expression of this gene is regulated by quorum sensing (QS) mechanism. Not activating gas vesicle genes during, for eample, an exponential growth phase enhances their grow rate. For QS, E. coli was transformed with two genes luxI and luxR . LuxI is an autoinducer synthase, which produces acyl-homoserin lactone (AHL). LuxR is a protein that binds to AHL. Since AHL freely diffuses though cell membrane, its concentration reflects density of the cells. When bound to AHL, LuxR stimulates Lux promoter of the gas vesicle gene. For gas vesicle proteins, only gvpA – C among at least 15 related genes are used to minimize the cost of the protein production.

        <a href="http://openwetware.org/images/8/89/Kyoto-Quorumgas.jpg" target="_blank"><img alt="Quorumgas" src="http://openwetware.org/images/8/89/Kyoto-Quorumgas.jpg" border="0" width="300" height="250" /></a>
  • Function C: Light-dependent flagella rotation

    • Our engineered E. coli is designed more likely to continue counter clockwise (CCW) flagella rotatation in the darkness due to cheZ.

    The mechanism consists of following four points:

    1. CCW is promoted in the presence of CheZ.
    2. In our E.coli, the synthesis of CheZ is accelerated in the darkness and is repressed in the light.
    3. Then, the number of E.coli rotating flagella CCW on the darker side of the object on which E.coli is binding exceeds that on the lighter side.
    4. The object on which E.coli is binding can gain force in the direction of lighter place.

    alternative below

    Our E. coli is designed to sense light and then generate thrust as following.

    • CCW rotation of flagella produces thrust
    • CCW is promoted in the presence of CheZ.
    • In our E.coli, cheZ expression is activated by light.

    For the production of light activated protein. three genes, ho1, pcyA, and cph8 of BioBrick parts were introduced. This protein is phosphorylated in light exposure and activates a specific promoter and expression of its downstream gene cheZ.

       <a href="http://openwetware.org/images/e/e2/Kyoto-Flagella.jpg" target="_blank"><img alt="Flagella" src="http://openwetware.org/images/e/e2/Kyoto-Flagella.jpg" border="0" width="300" height="250" /></a>

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