User:Lytao

From 2011.igem.org

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==<h1>Experiment results of the Basic Design <br/><br/>and Discussion</h1>==
 
-
<html ><a href= "#Verification of ΔcheZ  strain"><u>1.Verification of ΔcheZ  strain<html></u></a> <br/>
 
-
<html><a  href= "#Verification of the function of the constructed Aptamer-cheZ part">&nbsp;<u>2.Verification of the function of the constructed Aptamer-cheZ part</u></a></html><br/>
 
-
<html><a  href= "#Verification of the original Toggle-switch from PKU">&nbsp;<u>3.Verification of the original Toggle-switch from PKU<html></u> </a> <br/>
 
-
<html><a  href= "#Verification of the modified Toggle-switch">&nbsp;<u>4.Verification of the modified Toggle-switch</u> </a> </html><br/>
 
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<html><a  href= "#Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch">&nbsp;<u>5.Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch<html></u> </a><br/>
 
-
<html><a  href= "#Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch">&nbsp;<u>6.Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch</u> </a> </html><br/>
 
-
<html><a name="Verification of ΔcheZ  strain" id=""></a></html>
 
-
==Verification of ΔcheZ  strain==
+
<html lang="en">  
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;The size of colonies of ''E.coli'' strain RP1616 was much smaller than that ''E.coli'' steain RP437 under the same circumstance and after same period of incubation time(about 10h), and the result is shown in Figure1. In colony PCR using the primers of cheZ gene following , RP437 absolutely has much more outcomes than RP1616 and we conclude that RP1616 is actually a ΔcheZ strain.</p>
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<style type="text/css">
-
[[File:sst().jpg|center|350px| thumb|Figure1.]]
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<p align=center class="ppp">Figure1.The result of colony PCR(From left to right, the first lane is the marker, the third and the forth lane is the PCR outcome of strain RP437 and strain RP1616, the sixth and the seventh lane is the PCR outcome of strain RP437 and strain RP1616.)</p>
+
-
<html><a name="Verification of the function of the constructed Aptamer-cheZ part" id=""></a></html>
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==Verification of the function of the constructed Aptamer-cheZ part<html><a href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Aptamer-cheZ"><font size="4"><u>(protocol1)</u></font></a><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Dose-Dependent"><font size="4"><u>(protocol2)</u></font></a> </html>==
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<p>&nbsp;&nbsp;&nbsp;&nbsp;From the results shown in Figure2. We can be sure that the Aptamer-cheZ part actually works effectively, especially on 0.3% Semi-solid medium.</p>
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    color: #991133;
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[[File:X().jpg|center|650px| ]]
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    text-decoration: none;
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<p align=center class="ppp">Figure2. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Theophylline, Right:0.3%agar with 0.25mM Theophylline)</p>
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<html><a name="Verification of the original Toggle-switch from PKU" id=""></a></html>
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==Verification of the original Toggle-switch from PKU<html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch"><font size="4"><u>(protocol)</u></font></a> </html>==
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[[File:Res14.jpg|300px|center ]]
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<p align=center class="ppp">Figure3.A conlony of the bacteria with the original Toggle Switch exhibit two different states(4X Objective) </p>
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}
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<p>&nbsp;&nbsp;&nbsp;&nbsp;By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 8:25. It means the original Toggle Switch is not fit for our purpose, so we modify the original by luxPR-cI device.</p>
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-
<html><a name="Verification of the modified Toggle-switch" id=""></a></html>
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==Verification of the modified Toggle-switch  <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch"><font size="4"><u>(protocol)</u></font></a> </html>==
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    float: left;
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    padding: 30px 22px;
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    padding: 30px 28px 30px 15px;
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    padding: 30px 28px 30px 15px;
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[[File:Rs14.jpg|550px|center ]]
 
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<p align=center class="ppp">Figure4. The conlonies of the bacteria with the modified Toggle Switch exhibit two different states or just one state(4X Objective)</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 6:1. It is useful to our project design.</p>
 
-
<html><a name="Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch" id=""></a></html>
 
-
==Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch  <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Semi-Solid"><font size="4"><u>(protocol1)</u></font></a> <a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle"><font size="4"><u>(protocol2)</u></font></a></html>==
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/* end twitter */
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[[File:Resul.jpg|650px|center ]]
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<p align=center class="ppp">Figure5.The growing state of the reprogrammed bacteria with original Toggle-switch-Aptamer-cheZ Device(Red circle:the range of motion, Blue Triangle:the gradient of thepphylline)</p>
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-
[[File:Resddul.jpg|300px|center ]]
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-
<p align=center class="ppp">Figure6.The fluorescent Photo(4X Objective) of the colony shown in Figure5 Left.</p>
+
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;According to the results above, by using this device we can not make the reprogrammed bacteria be divided into two different states effectively, and because of the bias to the green fluorescence and moving towards the high concentration of the theophylline, the bacteria trending to stay are very rare.</p>
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<html><a name="Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch" id=""></a></html>
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==Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Toggle Switch-Aptamer-cheZ"><font size="4"><u>(protocol)</u></font></a> </html>==
 
-
[[File:Re.jpg|center|480px ]]
 
-
<p align=center class="ppp">Figure7.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure7 shows the fluctuation of expression of the modified Toggle Switch device, the number of bacteria with red fluorescent decreases from right to left.</p>
 
-
[[File:Re3.jpg|900px|center]]
 
-
<p align=center class="ppp">Figure8.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure8 shows four different growing states of the reprogrammed bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria(from the second tube（from right to left) shown in Figure7), and from left to right the concentration of the theophylline increases.</p>
 
-
[[File:Results12.jpg|450px|center ]]
 
-
<p align=center class="ppp">Figure9.The fluorescent Photo(4X Objective）of the colony1 in the fourth image of Figure8, and from left to right the concentration of the theophylline increases.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure9 clearly display the dividing line between the reprogrammed bacteria at two different states, and the bacteria mainly express GFP indeed moving towards the high concentration of theophylline. This means the modified Toggle-switch-Aptamer-cheZ actually work as our design.</p>
 
-
==Discussion==
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.col_list ul{
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<p>&nbsp;&nbsp;&nbsp;&nbsp;The experiment result mainly depend on the the random fluctuation of expression of the modified Toggle Switch device, so this device has a certain probability to switch from one state to the other, then it may lead to stopping of the moving bacteria like colony2(Figure10.).</P>
+
    list-style-type:none;
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[[File:R2.jpg|350px|center ]]
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    list-style-image:none;
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<p align=center class="ppp">Figure10.The fluorescent Photo(4X Objective）of the colony2 in the lower right of Figure8, and from left to right the concentration of the theophylline increases.</p>
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    background-image:none;
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;To weaken this randomness, we may use the quorum sensing to control the moving of the reprogrammed bacteria, and we construct a model to simulate this proposal by computer ([https://2011.igem.org/Team:USTC-China/Drylab/modeling '''Modeling''']).
+
 
 +
}
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.col_list li{
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    text-align:center;
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    font-family:"Comic Sans MS", cursive;
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    font-size: 1.6em;
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    padding: 5px 15px;
 +
   
 +
    background-color:transparent;
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}
 +
 
 +
 
 +
 
 +
/* TEAM PAGE */
 +
.bio {
 +
    clear:both;
 +
    padding-bottom: 20px;
 +
    border-bottom: 1px solid #ccc;
 +
}
 +
.bio img {
 +
    float:left;
 +
    margin-right: 10px;
 +
}
 +
 
 +
#videoiframe {
 +
    overflow: hidden;
 +
    margin-top: 20px;
 +
}
 +
 
 +
</style>
 +
</head>
 +
</html>
 +
 
 +
<html>
 +
<head>
 +
<!-- TODO: add pre/n buttons -->
 +
<script>
 +
$(document).ready(function(){
 +
  $('.bio').css('display','none');
 +
$('#yhlbio').fadeIn(500);
 +
  $('.col_list li').css('cursor','pointer');
 +
  $('.col_list li').hover(function() {
 +
    $(this).css('background-color','#91A3ED');
 +
    }, function() {
 +
    $(this).css('opacity','0.6');
 +
  });
 +
 
 +
  $('.col_list li').click(function () {
 +
    $('.bio').hide();
 +
    var name = $(this).attr('id');
 +
    $('#'+name+'bio').fadeIn(500);
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 +
  });
 +
});
 +
</script>
 +
</head>
 +
 
 +
<body>
 +
 
 +
<div id="col_nav">
 +
    <div class="col_list">
 +
        <h2>Weeks</h2>
 +
<ul>
 +
    <li id="yhl">Jun 28-July 2</li>
 +
    <li id="swl">July 3-July 9</li>
 +
    <li id="zlc">July 10-July 16</li>
 +
    <li id="fzz">July 17-July 23</li>
 +
    <li id="shl">July 24-July 30</li>
 +
    <li id="ml">July 31-Aug 6</li>
 +
    <li id="mps">Aug 7-Aug 13</li>
 +
            <li id="yfl">Aug 14-Aug 20</li>
 +
    <li id="dz">Aug 21-Aug 27</li>
 +
    <li id="lna">Aug 28-Sep 3</li>
 +
    <li id="jhp">Sep 4-Sep 10</li>
 +
    <li id="xlz">Sep 11-Sep 17</li>
 +
            <li id="wyy">Sep 18-Sep 24</il>
 +
    <li id="ytl">Sep 25-Oct 1</li>
 +
            <li id="nw">Oct 2-Oct 8</li>
 +
            <li id="yz">Oct 9-Oct 15</il>
 +
         
 +
</ul>
 +
    </div><!-- end undergrads -->
 +
</div>
 +
 
 +
 
 +
 
 +
<div id="col_left">
 +
 
 +
<div class="bio" id="yhlbio">
 +
<h1>Jun 28-July 2</h1>
 +
</html>
 +
2011.6.28
 +
----
 +
cultivate the bacteria of [http://partsregistry.org/Part:BBa_K557000 cheZ] deficiency(RP1616),and the Control group(RP437).
 +
check the resistibility of RP1616 and RP437
 +
result:both of the two groups are of none resistibility.
 +
members:Siwei Luo, Fangzhou Zhao, Zhilin Chen...
 +
 
 +
2011.6.29
 +
----
 +
check the motility of RP1616 strain and RP437 strain
 +
cultivate Top10 strain
 +
result: the bacterial plaque of RP1616 is smaller than that of RP437, implying that RP1616 is of [http://partsregistry.org/Part:BBa_K557000 cheZ] deficiency, causing the decreased motility of the bacteria.
 +
 
 +
 
 +
6.30
 +
----
 +
prepare for the competent cell of Top10 strain cultivated on 6.29
 +
extract the genome of Top10 strain and use it as complates to run PCR of CheZ
 +
result: the concentration of PCR result is too low to continue the experiments.
 +
 
 +
7.1
 +
----
 +
conduct PCR of CheZ again
 +
ligate the CheZ DNA segment to plasmid PSb1C3, and transform it into Top10 competent cells
 +
result:as the picture shows.
 +
 
 +
7.2
 +
----
 +
pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR
 +
result:The bacteria grow up well on dishes with chloromycetin resistency，verifying that the transformation is successfull
 +
while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.
 +
 
 +
 
 +
<html>
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="swlbio">
 +
<h1>July 3-July 9</h1>
 +
</html>
 +
7.3
 +
----
 +
extract the plasmids from the reproduced bacteria and send it to check the base sequence
 +
result: the concentration of the plasmids extracted is about 500ng/uL
 +
 
 +
7.4
 +
----
 +
extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2010 and put them into Top10 strain by transformation
 +
(LuxPr: Plate 2,24C  Terminator : Plate 1,6O)
 +
result: the bacteria on the plate with Amp resistency grows well
 +
7.5
 +
----
 +
pick up single colony from the Petri dish to reproduce in the liquid culture medium
 +
 
 +
 
 +
7.6
 +
----
 +
extract plasmids containing LuxPR and Terminator and make enzyme digestion with EcoR1 and Spe1 in order to validate the existence of the two parts
 +
recieve part Toggle Switch and rbs-Ci-ter from PKU and conduct transformation
 +
 +
result: Due to the short length of LuxPR and Terminator, the result of the enzyme digestion is hard to examine by electrophoresis.
 +
 
 +
7.7
 +
----
 +
result: there are a few colonies with part Toggle switch grown while no colony of rbs-Ci-ter appeared.
 +
 
 +
7.8
 +
----
 +
pick up single colony with part Toggle switch .
 +
extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2011 and put them into Top10 strain by transformation
 +
 
 +
7.9
 +
----
 +
pick up single colony with LuxPR & terminator.
 +
extract plasmids containing toggle switch to conduct PCR.
 +
 
 +
process PCR with plasmids aptamer-CheZ
 +
result:as the picture shows.
 +
the base sequence of CheZ extracted before is proved right.
 +
 
 +
 
 +
 
 +
<html>
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="zlcbio">
 +
<h1>July 10-July 16</h1>
 +
</html>
 +
7.10
 +
----
 +
ligate aptamer-CheZ with PSB1C3 plasmids
 +
 
 +
extract plasmids containing LuxPR and terminator
 +
result:the concentractions of plasmids LuxPR and terminator are both 600ng/ul
 +
 
 +
7.11
 +
----
 +
transform PSB1C3 containing aptamer-CheZ into bacteria and cultivate.
 +
7.12
 +
----
 +
carry out Double digestion of toggle switch and pSB1c3 and ligate them together.
 +
 
 +
pick up single colony from bacteria cultivated yesterday.
 +
 
 +
result: Both the location and the brightness of the electrophoretic bands correspond with our expectation.
 +
 
 +
7.14
 +
----
 +
transform the part rbs-ci-ter sent from PKU again into bacteria.
 +
check the colors of the colonies containing toggle switch.
 +
result: the ratio of red vs green is 8:25, verifying the function of toggle switch.
 +
 
 +
7.15
 +
----
 +
pick up the single colony from the plate yesterday and ten hours later extract the plasmids containing Ci-terminator.
 +
 
 +
 
 +
7.16
 +
----
 +
conduct PCR with the plasmids yesterday to reproduce rbs-ci-Term.
 +
result:failure
 +
 
 +
 
 +
<html>
 +
</div>
 +
 
 +
<div class="bio" id="fzzbio">
 +
<h1>July 17-July 23</h1>
 +
7.18-7.21
 +
----
 +
ligate the standard part Terminator to the end of toggle switch
 +
result: we conduct a enzyme digestion with EcoR1 and Pst1 to the final ligation product and the electrophoresis result shows that the ligation is successful.
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="shlbio">
 +
<h1>July 24-July 30</h1>
 +
7.22-7.29
 +
----
 +
perform the following experiments: PCR of rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.
 +
result: the concentration of plasmids with ligation gene is as high as ...
 +
 
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="mlbio">
 +
<h1>July 31-Aug 6</h1>
 +
7.30-8.3
 +
----
 +
PCR of LuxPR-rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.
 +
</div>
 +
 
 +
 
 +
<div class="bio" id="mpsbio">
 +
<h1>Aug 7-Aug 13</h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
 
 +
<div class="bio" id="yflbio">
 +
<h1>Aug 14-Aug 20</h1>
 +
 
 +
</div>
 +
 
 +
 
 +
<div class="bio" id="nwbio">
 +
<h1></h1>
 +
 
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="ytlbio">
 +
<h1></h1>
 +
 
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="dzbio">
 +
<h1></h1>
 +
 
 +
</div>
 +
 
 +
<div class="bio" id="lnabio">
 +
<h1></h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="jhpbio">
 +
<h1></h1>
 +
 
 +
 
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="yywbio">
 +
<h1></h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="xlzbio">
 +
<h1></h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 +
<div class="bio" id="yzbio">
 +
<h1></h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
 
 +
<div class="bio" id="wyybio">
 +
<h1></h1>
 +
 
 +
<div class="clear"></div>
 +
</div>
 +
</div>
 +
 
 +
</body>
 +
</html>

Latest revision as of 03:00, 29 October 2011


Weeks

  • Jun 28-July 2
  • July 3-July 9
  • July 10-July 16
  • July 17-July 23
  • July 24-July 30
  • July 31-Aug 6
  • Aug 7-Aug 13
  • Aug 14-Aug 20
  • Aug 21-Aug 27
  • Aug 28-Sep 3
  • Sep 4-Sep 10
  • Sep 11-Sep 17
  • Sep 18-Sep 24
  • Sep 25-Oct 1
  • Oct 2-Oct 8
  • Oct 9-Oct 15

Jun 28-July 2

2011.6.28

cultivate the bacteria of [http://partsregistry.org/Part:BBa_K557000 cheZ] deficiency(RP1616),and the Control group(RP437). check the resistibility of RP1616 and RP437 result:both of the two groups are of none resistibility. members:Siwei Luo, Fangzhou Zhao, Zhilin Chen...

2011.6.29

check the motility of RP1616 strain and RP437 strain cultivate Top10 strain result: the bacterial plaque of RP1616 is smaller than that of RP437, implying that RP1616 is of [http://partsregistry.org/Part:BBa_K557000 cheZ] deficiency, causing the decreased motility of the bacteria.


6.30

prepare for the competent cell of Top10 strain cultivated on 6.29 extract the genome of Top10 strain and use it as complates to run PCR of CheZ result: the concentration of PCR result is too low to continue the experiments.

7.1

conduct PCR of CheZ again ligate the CheZ DNA segment to plasmid PSb1C3, and transform it into Top10 competent cells result:as the picture shows.

7.2

pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR result:The bacteria grow up well on dishes with chloromycetin resistency，verifying that the transformation is successfull while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.


July 3-July 9

7.3

extract the plasmids from the reproduced bacteria and send it to check the base sequence result: the concentration of the plasmids extracted is about 500ng/uL

7.4

extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2010 and put them into Top10 strain by transformation (LuxPr: Plate 2,24C Terminator : Plate 1,6O) result: the bacteria on the plate with Amp resistency grows well 7.5

pick up single colony from the Petri dish to reproduce in the liquid culture medium


7.6

extract plasmids containing LuxPR and Terminator and make enzyme digestion with EcoR1 and Spe1 in order to validate the existence of the two parts recieve part Toggle Switch and rbs-Ci-ter from PKU and conduct transformation

result: Due to the short length of LuxPR and Terminator, the result of the enzyme digestion is hard to examine by electrophoresis.

7.7

result: there are a few colonies with part Toggle switch grown while no colony of rbs-Ci-ter appeared.

7.8

pick up single colony with part Toggle switch . extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2011 and put them into Top10 strain by transformation

7.9

pick up single colony with LuxPR & terminator. extract plasmids containing toggle switch to conduct PCR.

process PCR with plasmids aptamer-CheZ result:as the picture shows. the base sequence of CheZ extracted before is proved right.


July 10-July 16

7.10

ligate aptamer-CheZ with PSB1C3 plasmids

extract plasmids containing LuxPR and terminator result:the concentractions of plasmids LuxPR and terminator are both 600ng/ul

7.11

transform PSB1C3 containing aptamer-CheZ into bacteria and cultivate. 7.12

carry out Double digestion of toggle switch and pSB1c3 and ligate them together.

pick up single colony from bacteria cultivated yesterday.

result: Both the location and the brightness of the electrophoretic bands correspond with our expectation.

7.14

transform the part rbs-ci-ter sent from PKU again into bacteria. check the colors of the colonies containing toggle switch. result: the ratio of red vs green is 8:25, verifying the function of toggle switch.

7.15

pick up the single colony from the plate yesterday and ten hours later extract the plasmids containing Ci-terminator.


7.16

conduct PCR with the plasmids yesterday to reproduce rbs-ci-Term. result:failure


July 17-July 23

7.18-7.21 ---- ligate the standard part Terminator to the end of toggle switch result: we conduct a enzyme digestion with EcoR1 and Pst1 to the final ligation product and the electrophoresis result shows that the ligation is successful.

July 24-July 30

7.22-7.29 ---- perform the following experiments: PCR of rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction. result: the concentration of plasmids with ligation gene is as high as ...

July 31-Aug 6

7.30-8.3 ---- PCR of LuxPR-rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.

Aug 7-Aug 13

Aug 14-Aug 20

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