User:Lytao

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{{Team:USTC-China/temp2}}
{{Team:USTC-China/temp2}}
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== '''Artificial Innate Immunity System(AIIS)''' ==
 
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<html><a  href= "#Background">&nbsp;<u>1.Background</u> </a> </html><br/>
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<html lang="en">  
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<html><a  href= "#Purpose">&nbsp;<u>2.Purpose</u> </a> </html><br/>
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<style type="text/css">
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<html><a  href= "#Principle">&nbsp;<u>3.Principle</u> </a> </html><br/>
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<html><a  href= "#Theoretical Design">&nbsp;<u>4.Theoretical Design</u> </a> </html><br/>
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<html><a  href= "#Expermental Design and Results">&nbsp;<u>5.Expermental Design and Results</u> </a> </html><br/>
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<html><a  href= "#Conclusion and Discussion">&nbsp;<u>6.Conclusion and Discussion</u> </a> </html><br/>
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 +
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    color: #991133;
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    text-decoration: none;
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}
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a:hover {
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    text-decoration: underline;
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}
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.clear {
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    clear:both;
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}
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<html><a name="Background" id=""></a></html>
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#col_left{
 +
    float: left;
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    width: 550px;
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    padding: 30px 22px;
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    margin:0;
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}
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#col_right{
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    float: right;
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    width: 242px;
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    padding: 30px 28px 30px 15px;
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    margin:0;
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#col_nav{
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    float: left;
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    width: 242px;
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    padding: 30px 28px 30px 15px;
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    margin:0;
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}
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==Background==
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;From mid May to June 2011, enteropathogenic E.coli brings a big panic to many different European countries. Why such an ordinary bacteria as E.coli can lead to a clinical catastrophe and kill a lot of people? The answer may be complicate, but the most important reason is that we can not find out the pathogens rapidly and can not apply the effective treatments.To solve this problem, we try to use the normal E.coli, which keep the symbiotic relationship with us humanbeings, to be a safety weapon to defend ourselves against pathogens.</p>
 
-
[[File:At().jpg|center|350xp|thumb]]
 
-
<p align=center class="ppp">Figure1.Enteropathogenic ''Escherichia coli''(left) brings us a big panic(right,Frank Bellew, New York Daily Graphic, September 29, 1873)</p>
 
-
<html><a name="Purpose" id=""></a></html>
 
-
==Purpose==
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/* end twitter */
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Using reprogrammed intestinal bacteria as a safety weapon to destroy the pathogens which invade the intestinal mucosal system.</p>
+
 
-
<html><a name="Principle" id=""></a></html>
+
 
-
==Principle==
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.col_list ul{
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<p>&nbsp;&nbsp;&nbsp;&nbsp;As shown in Figure2, When pathogens invade the intestinal epithelium ,they will release some kind of chemical signal. After the reprogrammed intestinal bacteria capture this signal, such signal can activate the expression of the function gene ,then it will drive the reprogrammed intestinal bacteria to move toward the infection site from the original colony. Then the moving reprogrammed bacteria will express some lethal proteins at the infection site to kill pathogens by self-destruction, and the original colony will remain.</p>
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    list-style-type:none;
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<html><a name="Theoretical Design" id=""></a></html>
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    list-style-image:none;
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    background-image:none;
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}
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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;
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    background-color:transparent;
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}
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 +
 
 +
 
 +
/* TEAM PAGE */
 +
.bio {
 +
    clear:both;
 +
    padding-bottom: 20px;
 +
    border-bottom: 1px solid #ccc;
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}
 +
.bio img {
 +
    float:left;
 +
    margin-right: 10px;
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}
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 +
#videoiframe {
 +
    overflow: hidden;
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    margin-top: 20px;
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}
 +
 
 +
</style>
 +
</head>
 +
</html>
 +
 
<html>
<html>
<head>
<head>
 +
<!-- TODO: add pre/n buttons -->
 +
<script>
 +
$(document).ready(function(){
 +
  $('.bio').css('display','none');
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$('#yhlbio').fadeIn(500);
 +
  $('.col_list li').css('cursor','pointer');
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  $('.col_list li').hover(function() {
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    $(this).css('background-color','#91A3ED');
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    }, function() {
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    $(this).css('opacity','0.6');
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  });
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  $('.col_list li').click(function () {
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    $('.bio').hide();
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    var name = $(this).attr('id');
 +
    $('#'+name+'bio').fadeIn(500);
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 +
  });
 +
});
 +
</script>
</head>
</head>
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<div align="center">
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<object type="application/x-shockwave-flash" height=300 width=400 data="https://static.igem.org/mediawiki/2011/4/43/Application.swf" >
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<body>
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<param name="movie" value="https://static.igem.org/mediawiki/2011/4/43/Application.swf" />
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<param name="quality" value="high" />
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<div id="col_nav">
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<param name="" value="Exactfit"/>
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    <div class="col_list">
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</object>
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        <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>
 +
 +
 +
 
 +
<div id="col_left">
 +
 +
<div class="bio" id="yhlbio">
 +
<h1>Jun 28-July 2</h1>
</html>
</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...
-
==Theoretical Design==
+
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.
-
<p>'''1.AI(Auto Inducer）as a Chemical Signal'''</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;By using the device from the article ''Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen'' (Figure3.), we can modify our original design to achieve the function which we have described before.(As shown in Figure4.)</P>
 
-
[[File:v().jpg|center|380px|thumb]]
 
-
<p align=center class="ppp">Figure3.This image comes from  Nazanin Saeidi.etc (2011).</p>
 
-
[[File:q().jpg|center|560px|thumb]]
 
-
<p align=center class="ppp">Figure4.Theoretical design 1</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;In this design, the rhlPR is a RHLR/RHL Inducible Promoter, and we can assume the AI comes from pathogen is RHL. So the reprogrammed bacteria with green fluorescence will move toward the the infection site with a high concentration of RHL, while the reprogrammed bacteria with red fluorescence will stay at the original site waiting to creat the second wave of the reprogrammed bacteria with green fluorescence , then the reprogrammed bacteria with green fluorescence will decompose and release the pyosin to destroy the pathogens.</p>
 
-
<p>'''2.Other small molecules as chemical signals'''</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;By using the device from the article ''Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen'' (Figure 3.), we can modify our original design to achieve the function which we have described before(Figure 5.).</p>
 
-
[[File:j().jpg|center|560px|thumb]]
 
-
<p align=center class="ppp">Figure5.Theoretical design 2</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;In this design, we assume the small molecule is theophylline. So the reprogrammed bacteria with green fluorescence will move toward the infection site with a high concentration of theophylline and the expression of Lysis becomes more and more, while the reprogrammed bacteria with red fluorescence will stay at the original site and wait to creat the second wave of the reprogrammed bacteria with green fluorescence, they have been protected from being destroyed by the pyosin from the reprogrammed bacteria with green fluorescence. The reprogrammed bacteria with green fluorescence will destroy themselves and release the Pyosin, which is expressed at the beginning of the process, to kill the pathogens.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Besides, the Lysis protein can be replaced by ccdB protein, and the Pyosin also can be substituted by other proteins of destruction, and the theophylline also can be replaced by other small molecules or catalyzed from other molecules like caffein or amino acid.</p>
 
-
<html><a name="Expermental Design and Results" id=""></a></html>
 
-
==Simulated Experiment Design and Results <html><a  href= "https://2011.igem.org/Team:USTC-China/Wet_Lab/protocol#Artificial"><font size="4"><u>(protocol)</u></font></a> </html>==
+
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.
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;To put the original Toggle Switch-Aptamer-cheZ in use, we choose the second design above(As shown in Figure 5.) to be the simulated experiment construction(Figure 6.).</P>
+
7.2
-
[[File:g().jpg|center|560px|thumb]]
+
----
-
<p align=center class="ppp">Figure6.Simulated experiment construction</p>
+
pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;In this design, we use biobrick [http://partsregistry.org/Part:BBa_K117000 BBa_K117000] as the Lysis protein, and use biobrick [http://partsregistry.org/Part:BBa_K117001 BBa_K117001] to replace the pyosin. In which, the biobrick [http://partsregistry.org/Part:BBa_K117001 BBa_K117001] is ColE7-ImmE7 protein complex, and the lysis protein can remove Immunity protein from the complex with ColE7, then ColE7 regains its toxicity and ability to kill the EHEC bacteria or other bacteria nearby. Lysis protein itself can induce the lysis of host cell, exposing the interior cell contents and hence allows free diffusion of ColE7 proteins towards pathogenic bacteria strain.</P>
+
result:The bacteria grow up well on dishes with chloromycetin resistency，verifying that the transformation is successfull
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Under the consideration of bio-safety, we just use the ordinary E.coli strain with the chloramphenicol and tetracycline resistance as the target bacteia. After the experiment (details are shown in the Protocol), we have some reliable results shown below.</P>
+
while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.
-
[[File:nt().jpg|center|350px| thumb]]
+
-
<p align=center class="ppp">Figure7.The fluctuation of expression of the Toggle Switch device, the number of bacteria with red fluorescent decreases from tube1 to tube2.</p>
+
-
[[File:zzy().jpg|650px|center|thumb]]
+
-
<p align=center class="ppp">Figure8.The growing state of the target bacteria. Left(after 3h), Right(after 10h)</p>
+
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure8 shows the growing state of the target bacteria as the control of the experiment, in which the red circle represent the range of motion of the target, and from left to right the concentration of the theophylline increases.</P>
+
-
[[File:qpp().jpg|650px|center|thumb]]
+
-
<p align=center>Figure9.The growing state of the reprogrammed bacteria(colony1,from tube2) and the target bacteria(colony2). Left(after 3h), Right(after 10h)</p>
+
-
<P>&nbsp;&nbsp;&nbsp;&nbsp;Figure9 shows the growing state of the reprogrammed bacteria(from tube2) and the target bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria and the red circle represent the range of motion of the target bacteria, and from left to right the concentration of the theophylline increases.</p>
+
-
[[File:aaa().jpg|650px|center|thumb]]
+
-
<p align=center class="ppp">Figure10.The fluorescent Photo(4X Objective)of two colonies shown in Figure9, the reprogrammed bacteria(Left,from tube2) and the target bacteria(Right).</p>
+
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure10 shows two colonies, the colony of the reprogrammed bacteria which keeps the normal form, and the colony of the target bacteria which is deformed and has a hole as a plaque in the middle of it.</p>
+
-
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;[[File:ww().jpg|350px|center|thumb]]
+
-
<p align=center class="ppp">Figure11.The growing state of the reprogrammed bacteria(colony1,from tube3) and the target bacteria(colony2)(after 10h)</p>
+
-
<P>&nbsp;&nbsp;&nbsp;&nbsp;Figure11 shows the growing state of the reprogrammed bacteria(from tube3) and the target bacteria, in which the yellow circle represent the range of motion of reprogrammed bacteria and the red circle represent the range of motion of the target bacteria, and from left to right the concentration of the theophylline increases.</p>
+
-
[[File:ddd().jpg|650px|center|thumb]]
+
-
<p align=center class="ppp">Figure12.The fluorescent Photo(4X Objective) of two colonies shown in Figure11, the reprogrammed bacteria(Left,from tube2) and the target bacteria(Right).</p>
+
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure12 shows two colonies, the colony of the reprogrammed bacteria  and the colony of the target bacteria,both of them are deformed and have holes as plaques in the middle of the colony.</p>
+
-
<html><a name="Conclusion and Discussion" id=""></a></html>
+
-
==Conclusion and Discussion==
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Comparing with the control, the reprogrammed bacteria actually can destroy the target bacteria as the design requires and basically decrease the range of motion of the target bacteria.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;By measuring the range of motion of the control and the target bacteria dealed with the reprogrammed bacteria from different tubes(tube1, tube2, tube3, tube4), we get histogram below.</p>
 
-
[[File:wmm().jpg|500px|center|thumb]]
 
-
<p align=center class="ppp">Figure13.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Figure13 shows that the capability of eliminating the target bacteria grows with the number of bacteria with red fluorescen decreases. This result reveal a fact that the properties of the reprogrammed bacteria mainly depend on the random fluctuation of the expression of the Toggle Switch Device.This fact also explain the differences between the form of the colony1 from tube2(Figure10 left) and the colony1 from tube3(Figure12 Left).</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;Therefor to increase the controllability of this device, we can introduce the  quorum sensing into our design to make the reprogrammed bacteria destroy the target more effectively.</P>
 
-
==Future Work==
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;The clinical potential of the application of the synthetic biology is very huge. Such potential includes the development of the therapies for the treatment of infectious diseases and cancer, as well as approaches in vaccine development, microbiome engineering, cell therapy, and regenerative medicine.</p>
 
-
<p>&nbsp;&nbsp;&nbsp;&nbsp;With such background, our Toggle Switch-Aptamer-cheZ Device show us a new way to fight against different diseases.Using our analysis result about [https://2011.igem.org/Team:USTC-China/Drylab/riboswitch Riboswitch], we can standardize the riboswitches or aptamers as Plug-ins ,then our device is just like a kind of "weapon" platform, which uses different standard equipments, such as bacteriocin(just like our design), RNAi(shown in Figure14), or some other metabolites, to cure the diseases and keep us healthy.</p>
 
-
[[File:wdm().jpg|500px|center|thumb]]
 
-
<p align=center class="ppp">Figure14.This image comes from Warren C. Ruder.etc(2011)</p>
 
-
==References==
+
<html>
-
<p>1.Timothy S. Gardner,  Charles R. Cantor, James J. Collins (2000) Construction of a
+
<div class="clear"></div>
-
Genetic toggle switch in Escherichia coli. Nature, 403,339-342</p>
+
</div>
-
<p>2.Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules  and RNA. J.Am.Chem.Soc, 129,6870-6811</p>
+
 
-
<p>3.Chunbo Lou, Xili Liu, Ming Ni.etc (2010) Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Molecular Systems Biology, 6:350</p>
+
<div class="bio" id="swlbio">
-
<p>4.Joy Sinha, Samuel J Reyes, Justin P Gallivan (2010) Reprogramming bacteria to seek and destroy an herbicide. Nature Chemical Biology, 6,464-470</p>
+
<h1>July 3-July 9</h1>
-
<p>5.Steven L. Porter, George H. Wadhams, Judith P. Armitage (2011) Signal processing in complex chemotaxis pathways. Nature Reviews Microbiology, 9,153-165</p>
+
</html>
-
<p>6.Warren C. Ruder, Ting Lu, James J. Collins (2011) Synthetic Biology Moving into the Clinic. Science,333,1248-1252</p>
+
7.3
-
<p>7.Nazanin Saeidi, Choon Kit Wong, Tat-Ming Lo .etc (2011) Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Molecular Systems Biology,7:251</p>
+
----
 +
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>
 +
 
 +
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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

Retrieved from "http://2011.igem.org/User:Lytao"