Team:UST-Beijing/Notebook

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<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
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This is a template page. READ THESE INSTRUCTIONS.
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<div id="instructions" style="text-align: center; font-weight: normal; font-size: small; color: #f6f6f6; padding: 5px;">
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You are provided with this team page template with which to start the iGEM season.  You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki.  You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
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<div id="warning" style="text-align: center; font-weight: bold; font-size: small; color: #f6f6f6; padding: 5px;">
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You <strong>MUST</strong> have a team description page, a project abstract, a complete project description, a lab notebook, and a safety page.  PLEASE keep all of your pages within your teams namespace. 
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<br/>
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<p><h1>Project 2.</h1></p>
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<p><h1>Project I.</h1></p>
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<p><h2>Step 1：The construction of pSB1AC3/PR</h2></p>
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<p><h2>1.1：The construction of pSB1AC3（BBa_K603000）/PR</h2></p>
<table align="left" border="0" cellpadding="0" cellspacing="0" >
<table align="left" border="0" cellpadding="0" cellspacing="0" >
<tr>  
<tr>  
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<p>Cut pSB1AC3 w/EcoRI & SpeI</p>
<p>Cut pSB1AC3 w/EcoRI & SpeI</p>
<p>Combine the insert and vector with T4 ligase</p>
<p>Combine the insert and vector with T4 ligase</p>
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<p><h2>Step2: Gel electrophoresis</h2></p><br />
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<p><h2>1.2: Gel electrophoresis</h2></p><br />
<table align="left" border="0" cellpadding="5" cellspacing="5" >
<table align="left" border="0" cellpadding="5" cellspacing="5" >
<tr>  
<tr>  
<td> <img src="https://static.igem.org/mediawiki/2011/2/25/2pSB1AC3_EcoRI%26SpeI%E5%8F%8C%E9%85%B6%E5%88%87%E9%AA%8C%E8%AF%81.jpg" width="400" height="257"/> </td>
<td> <img src="https://static.igem.org/mediawiki/2011/2/25/2pSB1AC3_EcoRI%26SpeI%E5%8F%8C%E9%85%B6%E5%88%87%E9%AA%8C%E8%AF%81.jpg" width="400" height="257"/> </td>
<td width="220"> 1: wide range maker
<td width="220"> 1: wide range maker
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     <br/>2、3、4、5：the constructed plasmid cutted<br />
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     <br/>2、3、4、5：the constructed plasmid cutted
       with SpeI and EcoRI restriction enzymes
       with SpeI and EcoRI restriction enzymes
  </td>
  </td>
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<p>&nbsp;</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
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<p><h2>Step 3: DNA sequencing<h2></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p><h2>1.3: DNA sequencing</h2></p>
<img src="https://static.igem.org/mediawiki/2011/3/39/3%E6%B5%8B%E5%BA%8F%E7%BB%93%E6%9E%9C.PNG" width="640"/><br />
<img src="https://static.igem.org/mediawiki/2011/3/39/3%E6%B5%8B%E5%BA%8F%E7%BB%93%E6%9E%9C.PNG" width="640"/><br />
The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.
The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.
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<p>&nbsp;</p>
<p>&nbsp;</p>
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<p><h2>2.1 The  construction of pSG5/PR which is used for eukaryotic expression</h2>
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</p>
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<p><img src="https://static.igem.org/mediawiki/2011/c/c4/PR_for_pSG5.jpg" width="313" height="89" /><img src="https://static.igem.org/mediawiki/2011/f/fb/PSG5.JPG" width="321" height="231" /><img src="https://static.igem.org/mediawiki/2011/3/33/PSG5PR.jpg" width="321" height="244" /></p>
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<p>PR + pSG5 = pSG5/PR <br />
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  Cut PR w/EcoRI &amp; BamH I<br />
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  Cut pSG5 w/EcoRI &amp; BamH I<br />
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Combine the insert and vector with T4  ligase</p>
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<p><h2>2.2 Gel electrophoresis</h2></p>
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  <tr>
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    <td width="171"><p><img src="https://static.igem.org/mediawiki/2011/8/8b/%E5%A4%A7%E7%B4%AB_%E5%8F%8C%E5%88%87.jpg" alt="" width="147" height="221" /></p></td>
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    <td width="438"><p>1: wide range marker</p>
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    <p>2: the constructed plasmid pSG5/PR cut with EcoR1 and BamH1</p></td>
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  </tr>
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<p><h2>2.3  DNA sequencing</h2></p>
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<p><img src="https://static.igem.org/mediawiki/2011/1/15/2.3.PNG" width="650" height="412" /></p>
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<p>The result of DNA sequencing demonstrates  that there is no mutation in PR by comparing to the original  sequence.</p>
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<ol>
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</ol>
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<p><h2>3.1  The  construction of pBABE/PR</h2></p>
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<p><strong>1) PCR for amplifying more insert</strong><br />
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  Primer 1:         5'-ggg  gaa ttc taa acc acc atg ctt gcc-3'<br />
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  Primer 2:         5'-aaa  gaa ttc tca cta tta ggc gtt gct-3'<br />
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  <strong>Reaction  system:</strong><br />
 +
  10XPCR buffer                       5ul<br />
 +
  dNTP               4ul<br />
 +
  primer 1           1ul<br />
 +
  primer 2           2ul<br />
 +
  pfu                1ul<br />
 +
  template           1ul<br />
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  DMSO               5ul<br />
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  ddH2O              31ul<br />
 +
  Total              50ul<br />
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  <strong>Reaction  condition:</strong><br />
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  95℃       5min</p>
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<p>****************<br />
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  95℃       15s<br />
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  52℃       15s<br />
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  72℃       2min</p>
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<p>25cycles</p>
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<p>****************<br />
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  72℃       10min<br />
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4℃        ∞ </p>
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<strong>2) Ligation</strong></p>
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<p><img src="https://static.igem.org/mediawiki/2011/b/ba/PR.jpg" width="256" height="80" /><img src="https://static.igem.org/mediawiki/2011/a/a0/PBABE.jpg" width="355" height="289" /><img src="https://static.igem.org/mediawiki/2011/4/48/PBABEPR.jpg" width="295" height="266" /></p>
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<p>PR + pBABE = pBABE/PR <br />
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  Cut PR w/EcoRI <br />
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  Cut pBABE w/EcoRI <br />
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Combine the insert and vector with T4  ligase</p>
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<p><h2>3.2 Gel electrophoresis</h2></p>
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<p><img src="https://static.igem.org/mediawiki/2011/2/21/EcoR1%E9%AA%8C%E8%AF%81.jpg" width="162" height="248" /><img src="https://static.igem.org/mediawiki/2011/5/52/Sal1%E9%AA%8C%E8%AF%81.jpg" width="164" height="247" hspace="100" /></p>
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<p>1:Middle range maker</a>    　　　      1:DL2,000 DNA Marker</p>
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<p>2:cut with EcoR I　　　　　 2:cut with Sal I</p>
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<p>Figure 1 indicates that the insert is  combined with the vector successfully. <br />
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Figure 2 indicates that the insert is in  the right direction. </p>
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<p><h2>3.3 DNA sequencing</h2></p>
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<p><img src="https://static.igem.org/mediawiki/2011/2/21/3.3.jpg" width="558" height="349" /></p>
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<p>As before, the result of DNA sequencing demonstrates  that there is no mutation in PR by comparing to the original  sequence.</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<h2>4.1  The  construction of BBa_K603001</h2>
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<p><a href="https://static.igem.org/mediawiki/2011/f/fa/BBa_K603001.jpg"><img src="https://static.igem.org/mediawiki/2011/f/fa/BBa_K603001.jpg" width="920" height="466" border="0"></a></p>
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<p>fig.1 Proteorhodopsin(PR)digested at EcoRI and PstI and ligased to pSB1C3(Linearized plasmid backbone Pre-cut at EcoRI and PstI ends). </p>
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<h2>4.2 Gel electrophoresis</h2>
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<p><a href="https://static.igem.org/mediawiki/2011/8/8f/Cut_gel.jpg"><img src="https://static.igem.org/mediawiki/2011/8/8f/Cut_gel.jpg" border="0"></a>&nbsp;</p>
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<p>fig.2 In this fig,NO.1 is Wide range DNA marker,NO.2-4 are plasmid BBa_K603001 digested at EcoRI and PstI,demonstrate that the plasmid is correct. </p>
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<h1>Project II</h1>
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<h2>1.The construction of lacI-DBD-LXR-LBD </h2>
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<p><a href="https://static.igem.org/mediawiki/2011/c/c8/LacI_DBD_goujian.jpgborder="0"></a></p>
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<p align="left"> fig.1 Amino acid sequence of lacI-DBD-LXR-LBD chimeric protein,the DNA binding domain of lacI is boxed.</p>
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<p align="left">The DNA sequencing(bottom left)and the gel electrophoresis of the digested pET28(bottom right) both demostrate that </p>
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<p align="left">the construction of lacI-DBD-LXR-LBD is exactly what we designed before.</p>
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<h2 align="left">2.The gene worknet </h2>
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<p align="left"><a href="https://static.igem.org/mediawiki/2011/3/3e/Shuangzhilixitong.jpg"><img src="https://static.igem.org/mediawiki/2011/3/3e/Shuangzhilixitong.jpg" width="756" height="487" border="0"></a></p>
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<h2>3.The experiment</h2>
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<p>https://static.igem.org/mediawiki/2011/a/a5/Circuit.JPG</p>
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<p>fig.1 The gene circuit of the two-plasmid system. </p>
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<p>https://static.igem.org/mediawiki/2011/c/c5/IPTG%2BKAN_2.jpg</p>
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<p>fig.2 The key element that affect the RFP are IPTG,Kan and our compound.</p>
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<p>https://static.igem.org/mediawiki/2011/1/1d/Quxiantu.jpg" </p>
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<p>fig.3 We have tried five compounds,compound B,C,D and E can active the gene and express rfp.The pET28a is </p>
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<p>control that have no lacI-DBD-LXR-LBD gene. </p>
==Notebook==
==Notebook==
You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.
You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.

Latest revision as of 05:40, 29 September 2011



Project I.

1.1：The construction of pSB1AC3（BBa_K603000）/PR




 

 

 

 

 

 

 

 

PR + pSB1AC3 = pSB1AC3/PR (new part)

Cut PR w/EcoRI & SpeI

Cut pSB1AC3 w/EcoRI & SpeI

Combine the insert and vector with T4 ligase

1.2: Gel electrophoresis


1: wide range maker
2、3、4、5：the constructed plasmid cutted with SpeI and EcoRI restriction enzymes



 

 

 

 

 

 

 

 

 

 

1.3: DNA sequencing


The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.

 

2.1 The construction of pSG5/PR which is used for eukaryotic expression

PR + pSG5 = pSG5/PR
Cut PR w/EcoRI & BamH I
Cut pSG5 w/EcoRI & BamH I
Combine the insert and vector with T4 ligase

2.2 Gel electrophoresis

1: wide range marker

2: the constructed plasmid pSG5/PR cut with EcoR1 and BamH1

2.3 DNA sequencing

The result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

3.1  The construction of pBABE/PR

1) PCR for amplifying more insert
Primer 1:         5'-ggg gaa ttc taa acc acc atg ctt gcc-3'
Primer 2:         5'-aaa gaa ttc tca cta tta ggc gtt gct-3'
Reaction system:
10XPCR buffer      5ul
dNTP               4ul
primer 1           1ul
primer 2           2ul
pfu                1ul
template           1ul
DMSO               5ul
ddH2O              31ul
Total              50ul
Reaction condition:
95℃       5min

****************
95℃       15s
52℃       15s
72℃       2min

25cycles

****************
72℃       10min
4℃        ∞

2) Ligation

PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase

3.2 Gel electrophoresis

1:Middle range maker 　　　 1:DL2,000 DNA Marker

2:cut with EcoR I　　　　　 2:cut with Sal I

Figure 1 indicates that the insert is combined with the vector successfully.
Figure 2 indicates that the insert is in the right direction.

3.3 DNA sequencing

As before, the result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

 

 

4.1 The construction of BBa_K603001

fig.1 Proteorhodopsin(PR)digested at EcoRI and PstI and ligased to pSB1C3(Linearized plasmid backbone Pre-cut at EcoRI and PstI ends).

4.2 Gel electrophoresis

 

fig.2 In this fig,NO.1 is Wide range DNA marker,NO.2-4 are plasmid BBa_K603001 digested at EcoRI and PstI,demonstrate that the plasmid is correct.

Project II

1.The construction of lacI-DBD-LXR-LBD

fig.1 Amino acid sequence of lacI-DBD-LXR-LBD chimeric protein,the DNA binding domain of lacI is boxed.

The DNA sequencing(bottom left)and the gel electrophoresis of the digested pET28(bottom right) both demostrate that

the construction of lacI-DBD-LXR-LBD is exactly what we designed before.

2.The gene worknet

3.The experiment

Circuit.JPG

fig.1 The gene circuit of the two-plasmid system.

IPTG%2BKAN_2.jpg

fig.2 The key element that affect the RFP are IPTG,Kan and our compound.

Quxiantu.jpg"

fig.3 We have tried five compounds,compound B,C,D and E can active the gene and express rfp.The pET28a is

control that have no lacI-DBD-LXR-LBD gene.

Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Retrieved from "http://2011.igem.org/Team:UST-Beijing/Notebook"