Team:Hong Kong HKUST/notebook/mod4

From 2013.igem.org

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<body>
<body>
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          <a href="https://2013.igem.org/Main_Page"><img id="iGEM_Logo" src="https://static.igem.org/mediawiki/2013/4/46/Igem_qgem_logo.png"></a>
 +
         
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<a href="http://www.ust.hk/eng/index.htm"><img id="hkust_Logo" src="https://static.igem.org/mediawiki/2013/5/55/Hkust_logo.gif"></a>
 +
<ol class="pos_fixed">
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1">Module 1</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod2">Module 2</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod3">Module 3</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod4">Module 4</a></il>
 +
</ol>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<div id="cover"></div>
<div id="cover"></div>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/attribution">Attribution</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/acknowledge">Acknowledgement</a></li>
</ul>
</ul>
</li>
</li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/project">Project</a>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a>
<ul>
<ul>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modelling">Modelling</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/data">Data Page</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/datapage">Data Page</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Results</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li>
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">notebook</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">protocols</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">safety</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li>
 +
 
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Interviews">Interviews</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Presentation">Presentation</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/blog">Blog</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Videos">Videos</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/interview">Interviews</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Article">Article</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/article/genet">Article</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li>
</ul>
</ul>
</li>
</li>
</ul>
</ul>
 +
</div>
</div>
-
<br><br><br><br><br><br><br>
+
<br><br><br><br><br><br><br><br>
 +
<div id="title"><center><h3>Glyoxylate Shunt Module's Notebook</h3></center></div>
<div id="flight">
<div id="flight">
  <div id="satu" align="center"> <h1>June 2013</h1>
  <div id="satu" align="center"> <h1>June 2013</h1>
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       <div>
       <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Wet lab<br>
+
       <div class='content' align="left">
-
·      Inoculation of pBlueScript KS(+) for training<br>
+
<p><br>Design primers for <i>aceA</i>, <i>aceB</i>, MLS and EF-1alpha promoter extraction<br />
-
·      Autoclave basic materials<Br>
+
  Mini prepped pCMV/myc/mito<br />
-
·      Preparing LB<br>
+
  Extracted BW25113 genome<br />
-
·      LB-Ampicillin plates poured<br>
+
  PCR out <i>aceA</i> and <i>aceB</i> for Biobrick<br />
-
Dry lab<br>
+
  PCR out MLS for fuse with <i>aceA</i> and <i>aceB</i><br />
-
Protocols review
+
  PCR out <i>aceA</i> and <i>aceB</i> to fuse with MLS</p><br>
       </div>
       </div>
       </div>
       </div>
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  </div>
  </div>
</div>
</div>
-
<div id="satu" align="center"> <h1><center>July 2013</center></h1>
+
<div id="satu" align="center"> <h1>July 2013</h1>
    
    
   <div>
   <div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Transformed iDUET101a<br />
 +
  Mini prepped transformant and do digestion check<br />
 +
  Make glycerol stock out of iDUET101a<br />
 +
  Extract out pSB1C3 from <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> using gel extraction with XbaI and PstI<br />
 +
  Extract <i>E. coli</i> BW25113 genome</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
   </div>
   </div>
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   <div>
   <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Ligation of <i>aceA</i> and <i>aceB</i> with pSB1C3<br />
 +
  Transformed <i>aceA</i>-pSB1C3 and <i>aceB</i>-pSB1C3<br />
 +
  Screen out colonies for <i>aceA</i>-pSB1C3 and <i>aceB</i>-pSB1C3 and do digestion check<br />
 +
  <i>aceB</i>-pSB1C3 confirmed and proceed with sequencing<br />
 +
<i>aceB</i>-pSB1C3 partial sequence confirmed</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
   </div>
   </div>
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   <div>
   <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Extracted out more pSB1C3 from BBa_J04450<br />
 +
  PCR out <i>aceA</i> with standard prefix and suffix<br />
 +
  Digestion of <i>aceA</i> with XbaI and PstI<br />
 +
  Ligate with pSB1C3<br />
 +
  Screen colonies and do digestion check<br />
 +
  <i>aceA</i>-pSB1C3 confirmed and proceed with sequencing<br />
 +
  <i>aceA</i>-pSB1C3 partial sequence confirmed<br />
 +
  Help Chloe do digestion check for P<i>ef-1alpha</i>-pSB1C3</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
   </div>
   </div>
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   <div>
   <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Extraction of P<i>ef-1alpha</i> for Biobrick<br />
 +
  Extract out more pSB1C3 from BBa_J04450<br />
 +
  Digestion of P<i>ef-1alpha</i> with EcoRI and PstI<br />
 +
  Ligation of P<i>ef-1alpha</i> with pSB1C3<br />
 +
  Transformed P<i>ef-1alpha</i>-pSB1C3 construct<br />
 +
  Screen colonies and do digestion check<br />
 +
  Weird bands show up, do colony PCR to confirm whether P<i>ef-1alpha</i> is ligated<br />
 +
  Colony PCR showed that P<i>ef-1alpha</i> is there<br />
 +
  Colony PCR with VF2 and VR primers didn&rsquo;t give any result</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
   </div>  
   </div>  
-
  <div>
 
-
      <a href='#' class='head'>Week 5</a>
 
-
      <div class='content' align="left">Content 1
 
-
      </div>
 
-
  </div>
 
-
</div>
 
<div id="satu" align="center"> <h1>August 2013</h1>
<div id="satu" align="center"> <h1>August 2013</h1>
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<div>
<div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Practice Gibson Assembly using known DNA and Gibson assembly mix<br />
 +
Extract MLS, P<i>ef-1alpha</i>, <i>aceB</i> and polyadenylation sequence using PCR </p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Help Chloe do digestion check of pCMV-<i>aceA</i><br />
 +
  pCMV-<i>aceA</i> construct confirmed<br />
 +
  Redo making P<i>ef-1alpha</i>-pSB1C3 construct<br />
 +
  Create PMT construct for western blotting training<br />
 +
  PMT construct made proceed with western blotting:</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Build construct for P<i>ef-1alpha</i> characterization<br />
 +
  P<i>cmv</i> – GFP, P<i>ef-1alpha</i> – GFP construct, P<i>cmv</i> – mCherry, P<i>ef-1alpha</i> – mCherry constructs<br />
 +
  Individual DNA was extracted using PCR <br />
 +
  Assembled using Gibson Assembly</p>
 +
<p>&nbsp; </p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Troubleshooting for P<i>ef-1alpha</i> characterization constructs<br />
 +
  Use Gibson Assembly to make P<i>ef-1alpha</i> – pSB1C3 for submission</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
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     <div>
     <div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Give P<i>cmv</i>-<i>aceA</i> to group 1 to transfect <br />
 +
  P<i>ef-1alpha</i> – pSB1C3 constructed <br />
 +
  Digestion checks and sequenced<br />
 +
  Improvement of existing Biobrick: J176171 primer designs for J176171 to engineer with standard prefix in RFC10 and RFC25 formats<br />
 +
  Found that J176171 are heat unstable and degrades after denaturation</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Receive transfected HEK293 cell from Group 1<br />
 +
  Do western blotting with mouse anti c-myc antibody as primary antibody and anti-mouse HRP as secondary antibody<br />
 +
  Western blotting showed that AceA is expressed in HEK293 cell in contrast to the negative control<br />
 +
  Build construct for P<i>ef-1alpha</i> characterization: P<i>ef-1alpha</i> – GFP – polyadenylation sequence – pSB1C3 <br />
 +
  Improvement of existing Biobrick: J176171 primer designs to extract GFP with engineered complementary sequence of J176171</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p>Extract GFP in RFC10 and RFC25 format <br />
 +
  Heat sensitivity test of J176171 at temperatures range from 40 to 50 degrees Celsius for Gibson Assembly</p>
 +
<p>&nbsp;</p>
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Latest revision as of 23:15, 27 September 2013

  1. Notebook
  2. Module 1
  3. Module 2
  4. Module 3
  5. Module 4








Glyoxylate Shunt Module's Notebook

June 2013

Week 4


Design primers for aceA, aceB, MLS and EF-1alpha promoter extraction
Mini prepped pCMV/myc/mito
Extracted BW25113 genome
PCR out aceA and aceB for Biobrick
PCR out MLS for fuse with aceA and aceB
PCR out aceA and aceB to fuse with MLS


July 2013

Week 1

 

Transformed iDUET101a
Mini prepped transformant and do digestion check
Make glycerol stock out of iDUET101a
Extract out pSB1C3 from BBa_J04450 using gel extraction with XbaI and PstI
Extract E. coli BW25113 genome

 

Week 2

 

Ligation of aceA and aceB with pSB1C3
Transformed aceA-pSB1C3 and aceB-pSB1C3
Screen out colonies for aceA-pSB1C3 and aceB-pSB1C3 and do digestion check
aceB-pSB1C3 confirmed and proceed with sequencing
aceB-pSB1C3 partial sequence confirmed

 

Week 3

 

Extracted out more pSB1C3 from BBa_J04450
PCR out aceA with standard prefix and suffix
Digestion of aceA with XbaI and PstI
Ligate with pSB1C3
Screen colonies and do digestion check
aceA-pSB1C3 confirmed and proceed with sequencing
aceA-pSB1C3 partial sequence confirmed
Help Chloe do digestion check for Pef-1alpha-pSB1C3

 

Week 4

 

Extraction of Pef-1alpha for Biobrick
Extract out more pSB1C3 from BBa_J04450
Digestion of Pef-1alpha with EcoRI and PstI
Ligation of Pef-1alpha with pSB1C3
Transformed Pef-1alpha-pSB1C3 construct
Screen colonies and do digestion check
Weird bands show up, do colony PCR to confirm whether Pef-1alpha is ligated
Colony PCR showed that Pef-1alpha is there
Colony PCR with VF2 and VR primers didn’t give any result

 

August 2013

Week 1

 

Practice Gibson Assembly using known DNA and Gibson assembly mix
Extract MLS, Pef-1alpha, aceB and polyadenylation sequence using PCR

 

Week 2

 

Help Chloe do digestion check of pCMV-aceA
pCMV-aceA construct confirmed
Redo making Pef-1alpha-pSB1C3 construct
Create PMT construct for western blotting training
PMT construct made proceed with western blotting:

 

Week 3

 

Build construct for Pef-1alpha characterization
Pcmv – GFP, Pef-1alpha – GFP construct, Pcmv – mCherry, Pef-1alpha – mCherry constructs
Individual DNA was extracted using PCR
Assembled using Gibson Assembly

 

Week 4

 

Troubleshooting for Pef-1alpha characterization constructs
Use Gibson Assembly to make Pef-1alpha – pSB1C3 for submission

 

September 2013

Week 1

 

Give Pcmv-aceA to group 1 to transfect
Pef-1alpha – pSB1C3 constructed
Digestion checks and sequenced
Improvement of existing Biobrick: J176171 primer designs for J176171 to engineer with standard prefix in RFC10 and RFC25 formats
Found that J176171 are heat unstable and degrades after denaturation

 

Week 2

 

Receive transfected HEK293 cell from Group 1
Do western blotting with mouse anti c-myc antibody as primary antibody and anti-mouse HRP as secondary antibody
Western blotting showed that AceA is expressed in HEK293 cell in contrast to the negative control
Build construct for Pef-1alpha characterization: Pef-1alpha – GFP – polyadenylation sequence – pSB1C3
Improvement of existing Biobrick: J176171 primer designs to extract GFP with engineered complementary sequence of J176171

 

Week 3

 

Extract GFP in RFC10 and RFC25 format
Heat sensitivity test of J176171 at temperatures range from 40 to 50 degrees Celsius for Gibson Assembly

 

Week 4
Content 1
Week 5
Content 1

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