Team:NCTU Formosa/WetLab/Timer

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            <li><a href="https://2009.igem.org/Team:NCTU_Formosa/Team" target="_self"><b>Team</b></a>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/Team" target="_self">Team</a></li>
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<li><a href="http://life.nctu.edu.tw/~cptlab/Picasa%20HTML%20Exports/2009_iGEM-NCTU_Formosa/20091013.html" target="_blank"><b>Gallery</b></a>
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                <li><a href="http://life.nctu.edu.tw/~cptlab/Picasa%20HTML%20Exports/2009_iGEM-NCTU_Formosa/20091013.html" target="_blank">Gallery</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Mainworks" target="_self">Mainworks</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Labworks" target="_self">Lab works</a></li>
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<li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Groupworks" target="_self">Groupworks</a></li>
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                 <li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Timer" target="_self">Timer</a></li>
                 <li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Timer" target="_self">Timer</a></li>
                 <li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Counter" target="_self">Counter</a></li>
                 <li><a href="https://2009.igem.org/Team:NCTU_Formosa/WetLab/Counter" target="_self">Counter</a></li>
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<div id="innertext">
<div id="innertext">
      
      
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     <font size="3"><strong>Timer Function</strong><br></font>
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     <h4><strong>Timer Function</strong><br></h4>
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   <p>In our project, at first, we need a timer which can count different times we need. Base on this purpose, we decided to use lactose to repress the LacI(C0012) and make pLacI(R0011) express the down stream genes, cI434(C0052), tetR(C0040) and GFP(K145015) to lauch the timer function and control the duration of counting time. In addition, we plan to find out the different time we could control by using variant concentrations of lactose.</p><br><br><br><br>
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   <p>A controllable bio-timer which can count variable time duration was constructed in our bacterial referee. The timer function is controlled by the Plac promoter (<a href="http://partsregistry.org/Part:BBa_R0011" style="text-decoration:none;">BBa_R0011</a>), and the concentration of lactose determines timer’s working length. Lactose was used to repress the LacI protein (<a href="http://partsregistry.org/Part:BBa_C0012" style="text-decoration:none;">BBa_C0012</a>) and make the promoter Plac(BBa_R0011) express the down stream genes, cI434(<a href="http://partsregistry.org/Part:BBa_C0052" style="text-decoration:none;">BBa_C0052</a>), tetR(<a href="http://partsregistry.org/Part:BBa_C0040" style="text-decoration:none;">BBa_C0040</a>) and Green Fluorescent Protein (GFP)(<a href="http://partsregistry.org/Part:BBa_K145015" style="text-decoration:none;">BBa_K145015</a>) to implement the timer function. When added lactose is consumed by E. coli, the Red Fluorescent Protein (RFP) start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up and the product lose freshness. A key feature of the bio-timer is that make the longer timing function up to 8-20 hrs.</p><br><br><br>
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    <div id="innerimg" style="width:715px; height:470px;">
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  <div id="imgbox" style="width:700px; height:369px;">
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    <a href="https://2009.igem.org/Image:Timer_new01.png" ><img src="https://static.igem.org/mediawiki/2009/4/44/Timer_new01.png" border="0"></a>
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    </div>
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        </div>
      
      
      
      
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     <br><br><br><br>
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     <h4><strong>Component Descriptions</strong></h4><br>
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     <h4><strong>Component Descriptions</strong></h4><hr>
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<p>Device A
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<p>Strand A
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<ol>
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    <ol>
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<li>Constitutive promoter(J23106) always express the downstream genes, LacI and LuxR(C0062).</li>
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<li>Constitutive promoter (<a href="http://partsregistry.org/Part:BBa_J23106" style="text-decoration:none;">BBa_J23106</a>) always express the downstream genes, LacI and LuxR (<a href="http://partsregistry.org/Part:BBa_C0062" style="text-decoration:none;">BBa_C0062</a>).</li>
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<li>LacI inhibits the pLacI expression in the Device B(K188261).</li>
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<li>LacI inhibits the activity of promoter PLac in the strand B.</li>
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<li>LuxR combines to AHL to form the complex which induce the expression of pcIIp22(K145150) in Device F.</li>
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<li>Complex which combines LuxR with AHL induce the expression of pcIIp22 (<a href="http://partsregistry.org/Part:BBa_K145150" style="text-decoration:none;">BBa_K145150</a>) in strand F.</li>
     </ol>
     </ol>
     </p>
     </p>
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<p>Device B
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<p>Strand B
     <ol>
     <ol>
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<li>acI-regulated promoter (R0011) is to be inactive in the presence of LacI, and when lactose added binds LacI, the promoter will work.</li>
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<li>In the abscence of lactose, the LacI repressor binds to the promoter Plac(BBa_R0011) and prevents RNA polymerase bound to the promoter (Plac) from transcribing the dawnstream genes. When lactose enters the bacterium, it binds to the LacI repressor and causes a conformational change that inhibits its ability to bind DNA. Consequently, the promoter Plac can transcribe the daenstream genes.</li>
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<li>The cI repressor (C0052) inhibits the 434 cI-regulated promoter(R0052) in the Device E.</li>
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<li>The cI repressor (BBa_C0052) inhibits the 434 cI-regulated promoter PcI434 (BBa_R0052) in the strand E.</li>
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<li>Tetracycline repressor (C0040) inhibits the tetR-repressible promoter(R0040) in the Device C.</li>
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<li>Tetracycline repressor (BBa_C0040) inhibits the tetR-repressible promoter PtetR (BBa_R0040) in the strand C.</li>
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<li>Green fluorescent protein (K145015) expression makes us know that the Timer starts to count.</li>
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</ol>
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      </ol>
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     </p>
     </p>
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<br><br><br><br>
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<br><br><br>
      
      
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     <h4><Strong>Timer Mechanisms</Strong><br></h4>
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     <h4><Strong>Principle and Mechanism</Strong></h4><hr>
      
      
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<p>There are two phases during our timer is working:
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<p>The timer function in our bacterial referee has three stages:
      
      
     <ol>
     <ol>
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<li>Standby phase: the medium are colorless<br>
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<li>Standby phase: bacteria grow in lactose-free medium.</li>
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    <p>For our design, we use lactose as the repressor for this chemical timer. Before counting the time, lactose does not be added to the medium, we can know that LacI in Device A is expressed for repressing the promoter LacI in the Device B. In this condition, Device B does not work and also GFP‧LVA is not expressed. During this time, E.coli does nothing but grow and cells in the medium are colorless. </p></li>
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        <li> Lactose-accession phase: lactose is added in medium. </li>
 +
        <li> Lactose-consumption phase: bacteria eat all lactose, and the medium does not contain lactose. </li>
 +
        </ol>
 +
    </p>
      
      
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     <div id="innerimg" style="width:659px; height:245px;">
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   <div id="imgbox" style="width:646px; height:210px;">
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    <br><br>
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     <a href="https://2009.igem.org/Image:Timer01.png" ><img src="https://static.igem.org/mediawiki/2009/0/04/Timer01.png" border="0"></a>
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 +
    <p>
 +
    <ol>
 +
    <strong><li>Standby stage: timer not start yet, and the medium is colorless</strong>
 +
     <br><br><Br>
 +
        <div id="innerimg" style="width:465px; height:257px;">
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   <div id="imgbox" style="width:450px; height:237px;">
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     <a href="https://2009.igem.org/Image:Timer_new02.png" ><img src="https://static.igem.org/mediawiki/2009/2/29/Timer_new02.png" border="0"></a>
     </div>
     </div>
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    </div>
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        </div>
 +
       
 +
    <p>For our design, we use lactose as the activator for this bio-timer. When bacteria grow in lactose-free medium, the lacI (BBa_K091121) is produced by constitutive promoter and represses the Plac (<a href="http://partsregistry.org/Part:BBa_K091110" style="text-decoration:none;">BBa_K091110</a>) promoter. In this condition, strand B does not work and also GFP‧LVA is not expressed. During this time, E.coli does nothing but grow and cells in the medium are colorless.</p></li>
 +
    <strong><li>Lactose-accession phase: the medium is green </strong>
 +
    <p>While E.coli accumulates to the quantity we need (OD=0.1), we add lactose to start our bio-timer. After adding lactose, lactose removes the repression of LacI protein and LacI cannot repress the promoter PLac(BBa_K091110). When lactose is added in the medium, lactose binds with lacI (<a href="http://partsregistry.org/Part:BBa_K091121" style="text-decoration:none;">BBa_K091121</a>) repressor and releases the inhibition of B strand, so the PlacI (BBa_K091110)promoter starts transcripting GFP-LVA (BBa_K145015). Then, the medium are green in this stage. </p></li>
 +
    <strong><li>Lactose-consumption phase: the medium is red</strong>
 +
    <p>For our bio-timer, the concentration of lactose determines timer’s working length. We need the nature function in E.coli which is the lactose operon and the operons are inherent in E.coli. When lactose exists, the lactose operon is induced for translating the three enzymes, lacZ, lacY and lacA, which are for the degradation of lactose into glucose. To sum up, the bio-timer start to count time after lactose addition and stop working when lactose totally is metabolized because of the lactose operon. When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the medium turns from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up.</p></li>
 +
    </ol></p>
-
<li>Lactose-accession phase: the medium are green<br>
 
-
    <p>However, while E.coli accumulates to the quantity we need (OD=0.1), we add lactose to start our chemical timer. As a repressor, lactose removes the repression of LacI to promoter LacI. After adding lactose, LacI cannot repress the promoter LacI when the lactose exists. Promoter Lac switches on the downstream genes expression in this condtion. For our chemical timer, we need the nature function in E.coli which is the lactose operon and the operon is inherent in E.coli. When lactose exists, lactose operon will be induced for translating the three enzymes, lacZ, lacY and lacA, which are for the degradation of lactose into glucose. To sum up, the timer will start to count time after lactose addition and stop working when lactose totally is metabolized because of the lactose operon.</p></li>
 
      
      
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    <div id="innerimg" style="width:629px; height:245px;">
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        <br><br><br><br>
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  <div id="imgbox" style="width:614px; height:210px;">
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    <a href="https://2009.igem.org/Image:Timer02.pngg" ><img src="https://static.igem.org/mediawiki/2009/1/13/Timer02.png" border="0"></a>
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        <h4><strong>Present Achivements</strong></h4><hr>
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    </div>
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    </div>
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 +
        <p>The <strong> strand A (<a href="http://partsregistry.org/Part:BBa_K188161" style="text-decoration:none;">BBa_K188161</a>)</strong> always express the downstream genes, LacI and LuxR (<a href="http://partsregistry.org/Part:BBa_C0062" style="text-decoration:none;">BBa_C0062</a>) and <strong>strand B(<a href="http://partsregistry.org/Part:BBa_K188261" style="text-decoration:none;">BBa_K188261</a>)</strong> always express the downstream genes, LacI and LuxR (<a href="http://partsregistry.org/Part:BBa_C0062" style="text-decoration:none;">BBa_C0062</a>) were developed and sequenced. However, we do not have more time to keep on further experiments for testing. The experiments we plan to do are as follows:<br>Since lactose is used as the repressor for LacI protein, the carbon source in the broth can not be glucose or any carbon source will be the priority of use to lactose. Therefore, we choose the glycerol as our carbon source in M9 medium for the experiment.</p>
 +
       
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        <p>
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<br><br><br><br>
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        <ul>
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        <li>Growth curve determination
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<ol><li>The A and B strands are transformed to the host cells, and the cells are cultured in the M9 medium with glycerol as the carbon source to determine growth curve.</li><li>Contribution of the curve between time and O.D. (optical density).</li></ol></li>
-
<h4><strong>Present Achivements</strong></h4>
+
<li>Time-delay examination
 +
<ol><li>Base on the above growth curve, add the lactose to final concentration is 1mM (compared with IPTG induction concentration) when O.D. of culture arrive to 0.1.</li><li>Detect the intensity of green fluorescence after lactose is added. Draw the curve between the time and intensity of green fluorescence.</li></ol></li>
 +
        <li>Variant duration design
 +
        <ol><li>Follow the second experiment, use the different concentrations of lactose to find out the time duration we want.</li><li>Contribution of the model between the concentration of lactose and intensity of green fluorescence with time.</li></ol></li>
 +
        </ul>
 +
       
 +
       
 +
      <br><br><br><br>
 +
       
 +
        <h4><strong>Color distinguishing test</strong></h4><hr>
 +
   
 +
   
 +
        <p>Although we can not accomplish the experiments, we still made a color test to explain the possible result we expect. We cultured two tubes with host cells over night,and the host in one tube was transformed with <a href="http://partsregistry.org/Part:BBa_K145279" style="text-decoration:none;">BBa_K145279</a> and the other with pSB1A3. Over one night culture, the host transformed with BBa_K145279 displalyed obvious green (Fig.1 B); and the host transformed with pSB1A3 displalyed red (Fig.1 D)</p>
-
    <p>Until now, we have finished the construction of our Device A(K188161) and Device B and delivered them to be sequenced. Unfortunately, we do not have more time to keep on further experiments for testing. The experiments we plan to do are followings.<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Because we use lactose as the repressor for LacI, the carbon source in the broth can not be glucose or any carbon source will be the priority of use to lactose. Therefore, we choose the glycerol as our carbon source in M9 medium for the experiment.</p>
+
    <p>Bright green indicates the situation that bio-timer was launched. The red indicates that time’ s up. We assume that when in the transition state from green to red, the RFPs is produced and mix with GFPs, and the medium becomes yellow (Fig.1 C). Therefore,we mixed the tubes of GFPs and RFPs to represent the light-yellow or orange in the transition state. It’s very exciting that we can observe the change of color by our unaimed eyes.</p>
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 +
       
 +
        <br><br>
 +
       
 +
        <div id="innerimg" style="width:515px; height:380px;">
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  <div id="imgbox" style="width:500px; height:278px;">
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    <a href="https://2009.igem.org/Image:Timer_new03.png" ><img src="https://static.igem.org/mediawiki/2009/0/0d/Timer_new03.png" border="0"></a>
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    </div>
 +
            <div id="imgcaption">
 +
            <p>Fig. 1. The predicted spatiotemporal behavior of the bacterial referees. (A) The bacteria grow in colorless lactose-free medium. (B) When lactose is added in medium, lactose binds with lacI repressor and releases the inhibition of GFP. The medium become green in this stage. (C)When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. (D) When bacteria eat all lactose, the RFP is expressed by the Plux/cIIp22 promoter, the medium is red in this stage.</p>
 +
            </div>
 +
  </div>
-
<ul>
 
-
<li>Growth curve determination
 
-
<ol>
 
-
<li>The first and second devices are transformed to the host cells, and the cells are cultured in the M9 medium with glycerol as the carbon source to determine growth curve.</li>
 
-
<li>Contribution of the curve between time and O.D. (optical density).</li>
 
-
</ol>
 
-
</li>   
 
-
   
 
-
<li>Time-delay examination
 
-
<ol> 
 
-
    <li>Base on the above growth curve, add the lactose to final concentration is 1mM (compared with IPTG induction concentration) when O.D. of culture arrive to 0.1.</li>
 
-
    <li>Detect the green-fluorescent after lactose are added. Draw the curve between the time and intensity of green-fluorescent.</li>
 
-
    </ol>
 
-
</li>
 
-
<li>Variant duration design
 
-
<ol>
 
-
    <li>Follow the second experiment, use the different concentrations of lactose to find out the time we want.</li>
 
-
<li>Contribution of the model between the concentration of lactose and intensity of green-fluorescent with time.</li>
 
-
</ol>
 
-
</li>
 
-
</ul>
 
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    <br><br><br><br>
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<h4><strong>Timer Function Simulation</strong></h4>
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    <p>Based on the mechanism we designed for the timer, we made a simulation about the counting-time function with different concentrations of lactose. This simulation is constructed by four of six devices in our system. They are A,B,C and F (see the figure below). </p>
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    <div id="innerimg" style="width:538px; height:394px;">
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  <div id="imgbox" style="width:523px; height:369px;">
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    <a href="https://2009.igem.org/Image:Timer03.png" ><img src=" https://static.igem.org/mediawiki/2009/4/41/Timer03.png" border="0"></a>
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-
    </div>
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    </div>
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    <p>According to the diagrams, the simulations almost tally with our expectations. In figure A, the expressing intensity of GFP and the duration of timer functioning decrease with the concentration of lactose added decreasing. Moreover, the counting-time can be controlled from 20 hours to 8 hours. In the other figure B, the intensity of GFP is the same for different concentrations of AHL, but the expression of mRFP to the AHL-LuxR complex is quicker with higher concentration of AHL.</p>
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    <div id="innerimg" style="width:814px; height:640px; margin:30px 0px 30px 0px;">
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  <div id="imgbox" style="width:800px; height:574px;">
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    <a href="https://2009.igem.org/Image:Timer04.png" ><img src="https://static.igem.org/mediawiki/2009/3/31/Timer04.png" border="0"></a>
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    </div>
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    <div id="imgcaption">
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    <p>Figure A. The expressing intensity of GFP and the duration before mRFP expression decrease with lactose added decreases. The unit of [lactose] is not specific, and it’s an assumed amount.</p>
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    </div>
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    </div>
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    <div id="innerimg" style="width:814px; height:640px; margin:30px 0px 30px 0px;">
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  <div id="imgbox" style="width:800px; height:564px;">
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    <a href="https://2009.igem.org/Image:Timer05.png" ><img src=" https://static.igem.org/mediawiki/2009/f/f6/Timer05.png" border="0"></a>
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-
    </div>
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    <div id="imgcaption">
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    <p>Figure B.  The expression of mRFP to the AHL-LuxR complex is quicker with higher concentration of AHL. αout=outside [AHL], AHL is supposed to be the external harm bacteria.</p>
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    </div>
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    </div>
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    <br><br><br><br>
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-
<h4><strong>Color Distinguishing Test</strong></h4>
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-
 
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-
<p>Although we can not accomplish the experiments, we still made a little color test to explain the possible result we expect. We cultured two tubes with host cells over night,and the host in one tube was transformed with BBa_K145279 and the other with pSB1A3. Over one night, the color of tubes is very apparent and we can easily tell the colors. The images below are the tubes we cultured.</p>
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    <div id="innerimg" style="width:714px; height:270px; margin:30px 0px 30px 0px;">
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  <div id="imgbox" style="width:700px; height:220px;">
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    <a href="https://2009.igem.org/Image:Timer06.png" ><img src="https://static.igem.org/mediawiki/2009/6/66/Timer06.png" border="0"></a>
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    </div>
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    <div id="imgcaption">
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    <p>(Left) The green tube with the host transformed with BBa_K145279. (Middle) The red tube with the host transformed with pSB1A3.  (Right) From the left to the right: control tube(without inducer, K145279 tube, mixed tube, pSB1A3 tube. </p>
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  </div>
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    </div>
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<p>The green tube presented the situation that our timer was launched. The red one presented that the time was up. Then we assumed that when time is up, the red flurorenscent proteins would be produced and then mix with green flurorensent proteins.As a result, the tube would be yellow.</p>
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<p>Therefore,we mixed the two tubes by drops into a new tube, and we can see the color of the mixed tube is light-yellow or orange. It’s very exciting that we can just observe the change of color by our unaimed eyes.</p>
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Latest revision as of 17:31, 21 October 2009

Result - Timer


Timer Function

A controllable bio-timer which can count variable time duration was constructed in our bacterial referee. The timer function is controlled by the Plac promoter (BBa_R0011), and the concentration of lactose determines timer’s working length. Lactose was used to repress the LacI protein (BBa_C0012) and make the promoter Plac(BBa_R0011) express the down stream genes, cI434(BBa_C0052), tetR(BBa_C0040) and Green Fluorescent Protein (GFP)(BBa_K145015) to implement the timer function. When added lactose is consumed by E. coli, the Red Fluorescent Protein (RFP) start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up and the product lose freshness. A key feature of the bio-timer is that make the longer timing function up to 8-20 hrs.








Component Descriptions


Strand A

  1. Constitutive promoter (BBa_J23106) always express the downstream genes, LacI and LuxR (BBa_C0062).
  2. LacI inhibits the activity of promoter PLac in the strand B.
  3. Complex which combines LuxR with AHL induce the expression of pcIIp22 (BBa_K145150) in strand F.

Strand B

  1. In the abscence of lactose, the LacI repressor binds to the promoter Plac(BBa_R0011) and prevents RNA polymerase bound to the promoter (Plac) from transcribing the dawnstream genes. When lactose enters the bacterium, it binds to the LacI repressor and causes a conformational change that inhibits its ability to bind DNA. Consequently, the promoter Plac can transcribe the daenstream genes.
  2. The cI repressor (BBa_C0052) inhibits the 434 cI-regulated promoter PcI434 (BBa_R0052) in the strand E.
  3. Tetracycline repressor (BBa_C0040) inhibits the tetR-repressible promoter PtetR (BBa_R0040) in the strand C.




Principle and Mechanism


The timer function in our bacterial referee has three stages:

  1. Standby phase: bacteria grow in lactose-free medium.
  2. Lactose-accession phase: lactose is added in medium.
  3. Lactose-consumption phase: bacteria eat all lactose, and the medium does not contain lactose.



  1. Standby stage: timer not start yet, and the medium is colorless


    For our design, we use lactose as the activator for this bio-timer. When bacteria grow in lactose-free medium, the lacI (BBa_K091121) is produced by constitutive promoter and represses the Plac (BBa_K091110) promoter. In this condition, strand B does not work and also GFP‧LVA is not expressed. During this time, E.coli does nothing but grow and cells in the medium are colorless.

  2. Lactose-accession phase: the medium is green

    While E.coli accumulates to the quantity we need (OD=0.1), we add lactose to start our bio-timer. After adding lactose, lactose removes the repression of LacI protein and LacI cannot repress the promoter PLac(BBa_K091110). When lactose is added in the medium, lactose binds with lacI (BBa_K091121) repressor and releases the inhibition of B strand, so the PlacI (BBa_K091110)promoter starts transcripting GFP-LVA (BBa_K145015). Then, the medium are green in this stage.

  3. Lactose-consumption phase: the medium is red

    For our bio-timer, the concentration of lactose determines timer’s working length. We need the nature function in E.coli which is the lactose operon and the operons are inherent in E.coli. When lactose exists, the lactose operon is induced for translating the three enzymes, lacZ, lacY and lacA, which are for the degradation of lactose into glucose. To sum up, the bio-timer start to count time after lactose addition and stop working when lactose totally is metabolized because of the lactose operon. When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the medium turns from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up.





Present Achivements


The strand A (BBa_K188161) always express the downstream genes, LacI and LuxR (BBa_C0062) and strand B(BBa_K188261) always express the downstream genes, LacI and LuxR (BBa_C0062) were developed and sequenced. However, we do not have more time to keep on further experiments for testing. The experiments we plan to do are as follows:
Since lactose is used as the repressor for LacI protein, the carbon source in the broth can not be glucose or any carbon source will be the priority of use to lactose. Therefore, we choose the glycerol as our carbon source in M9 medium for the experiment.

  • Growth curve determination
    1. The A and B strands are transformed to the host cells, and the cells are cultured in the M9 medium with glycerol as the carbon source to determine growth curve.
    2. Contribution of the curve between time and O.D. (optical density).
  • Time-delay examination
    1. Base on the above growth curve, add the lactose to final concentration is 1mM (compared with IPTG induction concentration) when O.D. of culture arrive to 0.1.
    2. Detect the intensity of green fluorescence after lactose is added. Draw the curve between the time and intensity of green fluorescence.
  • Variant duration design
    1. Follow the second experiment, use the different concentrations of lactose to find out the time duration we want.
    2. Contribution of the model between the concentration of lactose and intensity of green fluorescence with time.




Color distinguishing test


Although we can not accomplish the experiments, we still made a color test to explain the possible result we expect. We cultured two tubes with host cells over night,and the host in one tube was transformed with BBa_K145279 and the other with pSB1A3. Over one night culture, the host transformed with BBa_K145279 displalyed obvious green (Fig.1 B); and the host transformed with pSB1A3 displalyed red (Fig.1 D)

Bright green indicates the situation that bio-timer was launched. The red indicates that time’ s up. We assume that when in the transition state from green to red, the RFPs is produced and mix with GFPs, and the medium becomes yellow (Fig.1 C). Therefore,we mixed the tubes of GFPs and RFPs to represent the light-yellow or orange in the transition state. It’s very exciting that we can observe the change of color by our unaimed eyes.



Fig. 1. The predicted spatiotemporal behavior of the bacterial referees. (A) The bacteria grow in colorless lactose-free medium. (B) When lactose is added in medium, lactose binds with lacI repressor and releases the inhibition of GFP. The medium become green in this stage. (C)When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. (D) When bacteria eat all lactose, the RFP is expressed by the Plux/cIIp22 promoter, the medium is red in this stage.