Team:ZJU-China/Notebook/August

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<div class="main">
<div class="main">
<div  class="pagetitle" id="nbook">
<div  class="pagetitle" id="nbook">
-
       <table style="background-color:transparent;" width="750" border="0" cellspacing="0" cellpadding="1">
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       <table style="background-color:transparent;" width="750" border="0" cellspacing="1" cellpadding="1">
   <tr>
   <tr>
     <td ><h3>Labnote</h3></td>
     <td ><h3>Labnote</h3></td>
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     <div id="leftul"><div class="leftul">  
     <div id="leftul"><div class="leftul">  
                 <div id="accordion">  
                 <div id="accordion">  
-
+
  <h4>May&June</h4>
-
<h4 class="current">July</h4>
+
<div class="pane" style="height:140px;"><a href="https://2011.igem.org/Team:ZJU-China/Notebook/Brainstorm">In the two months we discussed some new ideas and finally decided our project<br/>>> Click to see our brainstorm</a></div>
 +
<h4 >July</h4>
 
 
-
<div class="pane" style="display:block;">  
+
<div class="pane" >  
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook">In this month we......<br/>>>Click to see</a>
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook">In this month we......<br/>>>Click to see</a>
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</div>  
</div>  
-
<h4 >August</h4>  
+
<h4 class="current">August</h4>  
-
<div class="pane">  
+
<div class="pane" style="display:block;">  
-
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook">In this month we......<br/>>>Click to see</a>
+
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook/August">In this month we......<br/>>>Click to see</a>
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<div class="pane">  
<div class="pane">  
-
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook">In this month we......<br/>>>Click to see</a>
+
                 <a  href="https://2011.igem.org/Team:ZJU-China/Notebook/September">In this month we......<br/>>>Click to see</a>
</div>
</div>
-
+
<h4>Protocol</h4>
 +
        <div class="pane"><a href="https://2011.igem.org/Team:ZJU-China/Protocol">>>Click to see our lab protocol about biofilm formation</a></div>
  </div>
  </div>
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</div>
</div>
            
            
-
   <div class="bigblock">
+
   <div class="bigblock"><div class="inner" id="n_biobrick">
<div class="block" id="nsheet">  
<div class="block" id="nsheet">  
-
<h3>Week1</h3><hr/>  
+
<h3>Week5</h3><hr/>  
   
   
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
+
<table id="notesheet" width="650" border="1" cellspacing="1" cellpadding="1">  
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.4th Monday</td>  
+
     <td id="sheetleft">Aug.1st
-
     <td><table id="intable" width="328" border="0" cellspacing="0" cellpadding="1">  
+
Monday
 +
</td>  
 +
     <td><table id="intable" width="541" border="0" cellspacing="1" cellpadding="1">  
    
    
-
     <td width="128">• Aerobic cultivation of DH5α</td>  
+
     <td width="286">•Fusion PCR
 +
Vgb+YFP+tetR, NirB+RFP+tetR,
 +
</td>  
    
    
    
    
-
     <td width="196">• Preparation of apparatus for the formation of biofilm</td>  
+
     <td width="251" >•PCR: NirB, Vgb</td>  
    
    
</table></td>  
</table></td>  
   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul 5th Tuesday
+
     <td id="sheetleft">Aug.2nd
 +
Tuesday
 +
</td>  
</td>  
-
     <td>&nbsp;</td>  
+
     <td><table id="intable" width="615" border="0" cellspacing="1" cellpadding="1">
 +
 
 +
      <td width="140"> •Cut: 13K+10I, 22M+10I
 +
</td>
 +
 
 +
 
 +
    <td width="181" > • Purify: 13K+10I, 22M+10I<br/>
 +
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I
 +
</td>
 +
  <td width="142">• PCR backbones<br/>
 +
• Electrophresis
 +
</td>
 +
  <td width="144">• Gel excision and purification</td>
 +
</table></td>  
   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.6th
+
     <td id="sheetleft">Aug.3rd
Wednesday
Wednesday
 +
</td>  
</td>  
-
     <td><table width="217" border="0" cellspacing="0" cellpadding="1">  
+
     <td><table width="640" border="0" cellspacing="1" cellpadding="1">  
   <tr>  
   <tr>  
-
     <td width="215">•Receiving  primers ordered previously</td>  
+
     <td width="170">• Make Phusion Buffer, 5×isothermel buffer
-
      
+
</td>  
 +
     <td width="184">• colony PCR: 20H, 20J, 22B
 +
</td>
 +
   </tr>  
   </tr>  
</table>  
</table>  
Line 157: Line 182:
   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.7th
+
     <td id="sheetleft">Aug.4th
Thursday
Thursday
 +
</td>  
</td>  
-
     <td><table width="238" border="0" cellspacing="0" cellpadding="1">  
+
     <td><table width="618" border="0" cellspacing="1" cellpadding="1">  
   <tr>  
   <tr>  
-
     <td width="200">• Preparation of the aliquot of the primers</td>  
+
     <td >• Cut: 10I+22M, 10I+13; and purification
-
    <td width="200">• Something wrong with a shaking incubator</td>  
+
</td>  
 +
<td>• Ligation: Pvgb+22M+10I, Pnirb+13K+10I,<br/>
 +
• colony PCR: 13K+10I
 +
</td>  
 +
<td>• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C</td>
 +
 +
<td>• Transform: 1G, 3A, 5A, 7A from the distribution plate</td>
 +
   </tr>  
   </tr>  
</table>  
</table>  
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   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.8th
+
     <td id="sheetleft">Aug.5th
Friday
Friday
 +
</td>  
</td>  
-
     <td><table width="222" border="0" cellspacing="0" cellpadding="1">  
+
     <td><table width="627" border="0" cellspacing="1" cellpadding="1">  
       <tr>  
       <tr>  
-
        <td width="155">• Preparation of culture plates for the transformations</td>  
+
      <td >• Check the plates. Contamination, or no positive colonies</td>
 +
      <td>• Miniprep: 5 backbones, 22B-1, 22B-3</td>
 +
      <td>• PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result.</td>
 +
      <td>• Culture the positive colony.</td>  
       </tr>  
       </tr>  
     </table></td>  
     </table></td>  
   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.9th
+
     <td id="sheetleft">Aug.6th
Saturday
Saturday
 +
</td>  
</td>  
-
     <td><table width="313" border="0" cellspacing="0" cellpadding="1">  
+
     <td><table width="620" border="0" cellspacing="1" cellpadding="1">  
   <tr>  
   <tr>  
-
     <td width="127">• Preparation of culture plates for the transformations</td>
+
      
-
    <td width="182">• protocols of transformation</td>
+
   </tr>  
   </tr>  
</table>  
</table>  
Line 191: Line 229:
   </tr>  
   </tr>  
   <tr>  
   <tr>  
-
     <td id="sheetleft">Jul.10th
+
     <td id="sheetleft">Aug.7th
Sunday
Sunday
</td>  
</td>  
-
     <td><table width="246" border="0" cellspacing="0" cellpadding="1">  
+
     <td><table width="620" border="0" cellspacing="1" cellpadding="1">  
       <tr>  
       <tr>  
-
         <td>• Several colonies were picked up and cultivated in 5mL LB medium.
+
         <td width="244">• Cut: 22M,13K with E &amp; S
-
•Cryosectioning of biofilm
+
   
 +
</td>
 +
<td width="164">• Culture the red colonies from plate of pSB1C3</td>
 +
<td>• PCR: 5 backbones<br/>
 +
• Cut the PCR products with P+E and run the gel to confirm.
</td>  
</td>  
       </tr>  
       </tr>  
Line 204: Line 246:
   
   
</table>  
</table>  
-
<p>&nbsp;</p>  
+
<p>&nbsp;</p>  
</div>  
</div>  
 +
 +
<div class="block" id="nsheet">
 +
<h3>Week6</h3><hr/>
 +
 +
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
 +
  <tr><td width="76">Day</td><td width="349">Note</td></tr>
 +
 
 +
  <tr>
 +
    <td id="sheetleft">Aut.8th
 +
Monday
 +
 +
</td>
 +
    <td><table id="intable" width="100%" border="0" cellspacing="0" cellpadding="1">
 +
 
 +
    <td width="191">• The gel results of 5 backbones are right.
 +
</td>
 +
 
 +
 
 +
    <td width="249" >• Miniprep: pSB1C3, vgb+22M+10I</td><br />
 +
<td width="157">• Culture vgb+22M+1oI in hypoxia<br/>
 +
•PCR pSB1C3
 +
</td>
 +
 
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.9th
 +
Tuesday
 +
 +
 +
</td>
 +
    <td><table id="intable" width="100%" border="0" cellspacing="0" cellpadding="1">
 +
 
 +
      <td width="33%"> • Run the PCR product
 +
</td>
 +
 
 +
 
 +
    <td width="33%" > • PCR pSB1C3 again
 +
</td>
 +
  <td width="33%">• Run the product, results are good.
 +
</td>
 +
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.10th
 +
Wednesday
 +
 +
 +
</td>
 +
    <td><table width="100%" border="0" cellspacing="0" cellpadding="1">
 +
  <tr>
 +
    <td width="170">• Culture: 20H, 20J<br/>
 +
• Transform 13K from plate 3
 +
 +
</td>
 +
    <td width="184">• Miniprep: 20H-1, 20J-1
 +
</td>
 +
<td>• Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P</td>
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.11th
 +
Thursday
 +
 +
 +
</td>
 +
    <td><table width="100%" border="0" cellspacing="0" cellpadding="1">
 +
  <tr>
 +
    <td >• Run the digestion results. Bands are confirmed right.
 +
</td>
 +
<td>• Colony PCR vgb+22M+10I
 +
</td>
 +
<td>• Cut the plasmid of vgb+22M+10I
 +
 +
</td>
 +
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.12th
 +
Friday
 +
 +
 +
</td>
 +
    <td><table width="100%" border="0" cellspacing="0" cellpadding="1">
 +
      <tr>
 +
      <td >• Make new stock of competent cells</td>
 +
      <td>• Ligation: 13K+10I<br/>
 +
• Cut the PCR results and 22M with E+P
 +
</td>
 +
      <td>• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B</td>
 +
      <td>• Transform 13K+10I+pSB1K3<br/>
 +
• Cut 13K to validate. Results are right.
 +
</td>
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.13th
 +
Saturday
 +
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="0" cellpadding="1">
 +
  <tr>
 +
    <td>• Colony PCR: 13K+10I+pSB1K3</td>
 +
    <td>• Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath).</td>
 +
    <td>• Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,</td>
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.14th
 +
Sunday
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="0" cellpadding="1">
 +
      <tr>
 +
        <td width="244">• Transform: the  Gibson assembly results.
 +
 +
</td>
 +
<td width="164">• Miniprep: 13K+10I<br/>
 +
• Cut: 13K+10I
 +
</td>
 +
<td>• Purification: the digestion results.<br/>
 +
• Ligation: nirB+13K+10I
 +
 +
</td>
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
 +
</table>
 +
<p>&nbsp;</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>Week7</h3><hr/>
 +
<table id="notesheet" width="650" border="1" cellspacing="1" cellpadding="1">
 +
  <tr><td width="76">Day</td><td width="349">Note</td></tr>
 +
 
 +
  <tr>
 +
    <td id="sheetleft">Aug.15th
 +
Monday
 +
 +
 +
</td>
 +
    <td><table id="intable" width="603" border="0" cellspacing="1" cellpadding="1">
 +
 
 +
    <td width="191">• Plate: nirB+13K+10I
 +
</td>
 +
 
 +
 
 +
    <td width="249" >• Colony PCR: vgb+YFP+tetR +terminater</td><br />
 +
<td width="157">• Gibson PCR
 +
</td>
 +
 
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.16th
 +
Tuesday
 +
 +
 +
 +
</td>
 +
    <td><table id="intable" width="615" border="0" cellspacing="1" cellpadding="1">
 +
 
 +
      <td width="140"> • Colony PCR: vgb+22M+10I, nirB+13K+10I
 +
</td>
 +
 
 +
 
 +
    <td width="181" > • No bands of 13K; 22M confirmed
 +
</td>
 +
 +
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aut.17th
 +
Wednesday
 +
 +
 +
 +
</td>
 +
    <td><table width="640" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
    <td width="170">• Miniprep: 22M<br/>
 +
• Cut: 22M
 +
 +
 +
</td>
 +
    <td width="184">• Check CFP expression. No fluorescence.
 +
</td>
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.18th
 +
Thursday
 +
 +
 +
 +
</td>
 +
    <td><table width="618" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
    <td >• Transform: 13K, 10I, 22M, 12I, 18P
 +
</td>
 +
 +
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.19th
 +
Friday
 +
 +
 +
 +
</td>
 +
    <td><table width="627" border="0" cellspacing="1" cellpadding="1">
 +
      <tr>
 +
      <td >• Miniprep: 13K, 10I, 22M, 12I, 18P </td>
 +
      <td>• Cut: 13K, 10I, 22M, 12I, 18P
 +
</td>
 +
      <td>• Run the gel</td>
 +
   
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.20th
 +
Saturday
 +
 +
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
    <td>• Ligate 13K+10I, 22M+10I</td>
 +
 
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.21st
 +
Sunday
 +
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="1" cellpadding="1">
 +
      <tr>
 +
        <td width="244">• Transform: the  Gibson assembly results.
 +
 +
</td>
 +
<td width="164">• Miniprep: 13K+10I<br/>
 +
• Cut: 13K+10I
 +
</td>
 +
<td>• Purification: the digestion results.<br/>
 +
• Ligation: nirB+13K+10I
 +
 +
</td>
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
 +
</table>
 +
<p>&nbsp;</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>Week8</h3><hr/>
 +
 +
<table id="notesheet" width="650" border="1" cellspacing="1" cellpadding="1">
 +
  <tr><td width="76">Day</td><td width="349">Note</td></tr>
 +
 
 +
  <tr>
 +
    <td id="sheetleft">Aug. 22nd
 +
Monday
 +
 +
 +
 +
</td>
 +
    <td><table id="intable" width="603" border="0" cellspacing="1" cellpadding="1">
 +
 
 +
    <td width="191">• Transform 13K+10I, 22M+10I
 +
</td>
 +
 
 +
 
 +
    <td width="249" >• Test new primers.<br />
 +
• PCR: 22M+10I, 13K+10I
 +
</td>
 +
<td width="157">• Run the PCR products.<br />
 +
• Cut the PCR products.
 +
 +
</td>
 +
 
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug. 23rd
 +
Tuesday
 +
 +
 +
 +
 +
</td>
 +
    <td><table id="intable" width="615" border="0" cellspacing="1" cellpadding="1">
 +
 
 +
      <td width="140">• Test primers: CS/CP, VF/VR, pSB1_f/r
 +
</td>
 +
 
 +
 
 +
    <td width="181" > • Cut: 11P, 1F, 22M<br/>
 +
• Run the cut results.1F-1/2/3 are right.
 +
</td>
 +
<td>• Mix the 1F-1/2 purification products</td>
 +
<td>• Cut: 1F-1+1F-2(E+S), 22M-3(X+P), pSB1C3(E+P)</td>
 +
 +
</table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug. 24th
 +
Wednesday
 +
 +
 +
 +
 +
</td>
 +
    <td><table width="640" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
 
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug. 25th
 +
Thursday
 +
 +
 +
 +
 +
</td>
 +
    <td><table width="618" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
 
 +
 +
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug. 26th
 +
Friday
 +
 +
 +
</td>
 +
    <td><table width="627" border="0" cellspacing="1" cellpadding="1">
 +
      <tr>
 +
      <td >• Amplify: vgb, 1C3</td>
 +
     
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.27th
 +
Saturday
 +
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="1" cellpadding="1">
 +
  <tr>
 +
    <td>• Digest: Vgb, 1C3, fdfhF, 22M+10I, 13K+10I</td>
 +
 
 +
 +
  </tr>
 +
</table>
 +
</td>
 +
  </tr>
 +
  <tr>
 +
    <td id="sheetleft">Aug.28th
 +
Sunday
 +
 +
</td>
 +
    <td><table width="620" border="0" cellspacing="1" cellpadding="1">
 +
      <tr>
 +
        <td width="244">• Ligate: vgb+22M+10I, fdfhF+13K+10I
 +
 +
</td>
 +
<td width="164">• Transform the ligation results.
 +
</td>
 +
 +
      </tr>
 +
    </table></td>
 +
  </tr>
 +
 +
</table>
 +
<p>&nbsp;</p>
 +
</div>
 +
</div>
<script type="text/javascript">
<script type="text/javascript">
</script>
</script>
 +
<div class="inner" id="biofilm_jul">
 +
<div class="block" id="nsheet">
 +
<h3>1st August</h3><hr/>
-
 
+
<p>Freeze slicing of about 50μm. Observe under natural light microscope and can see red
 +
florescence. The thickness is about 130μm</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>4th August</h3><hr/>
 +
<p>Repeat the last experiment with silicone tube. 6rpm/s results in a flowing speed of 68ml/day</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>6th August</h3><hr/>
 +
<p>Similar to the last time. Did not use freeze slicing.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>15th August</h3><hr/>
 +
<p>Cultured E.coli in 5ml LB for 12h.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>16th August</h3><hr/>
 +
<p>Substituting a part of silicone tube with glass tube in silicone tube biofilm formation sets. Culture in
 +
37℃</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>17th August</h3><hr/>
 +
<p>Add 50ml LB and culture with circular culture.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>18th August</h3><hr>
 +
<p>12.00 found one free-flow pump stopped. Cooled for 15 minutes and turned on again. Could be
 +
over heated. Can see some bacteria on the bottom of the vessel and tube. When the pump started
 +
again, the bacteria was washed away.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>19th August</h3><hr/>
 +
<p>Can observe obvious white biofilm where the silicone tube joins the tube but cannot see red
 +
florescence, suspect contamination. No biofilm is observed on the glass tube under microscope.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>21st, August</h3><hr/>
 +
<p>Biofilm formation with large test tubes. Inoculate with 1% e.coli. Rubber tubes are attached to air
 +
pump with filter between air pump and the tube to prevent entrance of germs. Two sets use MSM
 +
+glucose medium and two sets use LB.<br/>
 +
Retry with glass tube biofilm formation set.</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h3>24th, August</h3><hr/>
 +
<p>Terminate biofilm formation, the glass slide left in the drawer to dry. Can see obvious rod like
 +
structure under 400 magnification. (spheres in MSM+glucose culture) No red florescence is seen.
 +
Most of the surface is covered by single layer cells but some part of it has thick bump like
 +
structure.<br/>
 +
No biofilm observed on the glass tube.</p>
 +
</div>
 +
</div>
 +
<script type="text/javascript">
 +
document.getElementById('p_intro').onclick= function(){
 +
document.getElementById('biofilm_jul').style.display='none'
 +
document.getElementById('n_biobrick').style.display='block'}
 +
document.getElementById('p_model').onclick= function(){
 +
document.getElementById('n_biobrick').style.display='none'
 +
document.getElementById('biofilm_jul').style.display='block'}
 +
</script>
</div>
</div>

Latest revision as of 14:51, 3 October 2011

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

Labnote

 Biobrick group | Biofilm group 

This group...........

Week5

DayNote
Aug.1st Monday
•Fusion PCR Vgb+YFP+tetR, NirB+RFP+tetR, •PCR: NirB, Vgb
Aug.2nd Tuesday
•Cut: 13K+10I, 22M+10I • Purify: 13K+10I, 22M+10I
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I 		• PCR backbones
• Electrophresis 		• Gel excision and purification
Aug.3rd Wednesday
• Make Phusion Buffer, 5×isothermel buffer • colony PCR: 20H, 20J, 22B
Aug.4th Thursday
• Cut: 10I+22M, 10I+13; and purification • Ligation: Pvgb+22M+10I, Pnirb+13K+10I,
• colony PCR: 13K+10I 		• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C • Transform: 1G, 3A, 5A, 7A from the distribution plate
Aug.5th Friday
• Check the plates. Contamination, or no positive colonies • Miniprep: 5 backbones, 22B-1, 22B-3 • PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result. • Culture the positive colony.
Aug.6th Saturday
Aug.7th Sunday
• Cut: 22M,13K with E & S • Culture the red colonies from plate of pSB1C3 • PCR: 5 backbones
• Cut the PCR products with P+E and run the gel to confirm.

 

Week6

DayNote
Aut.8th Monday
• The gel results of 5 backbones are right. • Miniprep: pSB1C3, vgb+22M+10I • Culture vgb+22M+1oI in hypoxia
•PCR pSB1C3
Aug.9th Tuesday
• Run the PCR product • PCR pSB1C3 again • Run the product, results are good.
Aug.10th Wednesday
• Culture: 20H, 20J
• Transform 13K from plate 3 		• Miniprep: 20H-1, 20J-1 • Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P
Aug.11th Thursday
• Run the digestion results. Bands are confirmed right. • Colony PCR vgb+22M+10I • Cut the plasmid of vgb+22M+10I
Aug.12th Friday
• Make new stock of competent cells • Ligation: 13K+10I
• Cut the PCR results and 22M with E+P 		• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B • Transform 13K+10I+pSB1K3
• Cut 13K to validate. Results are right.
Aug.13th Saturday
• Colony PCR: 13K+10I+pSB1K3 • Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath). • Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,
Aug.14th Sunday
• Transform: the Gibson assembly results. • Miniprep: 13K+10I
• Cut: 13K+10I 		• Purification: the digestion results.
• Ligation: nirB+13K+10I

 

Week7

DayNote
Aug.15th Monday
• Plate: nirB+13K+10I • Colony PCR: vgb+YFP+tetR +terminater • Gibson PCR
Aug.16th Tuesday
• Colony PCR: vgb+22M+10I, nirB+13K+10I • No bands of 13K; 22M confirmed
Aut.17th Wednesday
• Miniprep: 22M
• Cut: 22M 		• Check CFP expression. No fluorescence.
Aug.18th Thursday
• Transform: 13K, 10I, 22M, 12I, 18P
Aug.19th Friday
• Miniprep: 13K, 10I, 22M, 12I, 18P • Cut: 13K, 10I, 22M, 12I, 18P • Run the gel
Aug.20th Saturday
• Ligate 13K+10I, 22M+10I
Aug.21st Sunday
• Transform: the Gibson assembly results. • Miniprep: 13K+10I
• Cut: 13K+10I 		• Purification: the digestion results.
• Ligation: nirB+13K+10I

 

Week8

DayNote
Aug. 22nd Monday
• Transform 13K+10I, 22M+10I • Test new primers.
• PCR: 22M+10I, 13K+10I 		• Run the PCR products.
• Cut the PCR products.
Aug. 23rd Tuesday
• Test primers: CS/CP, VF/VR, pSB1_f/r • Cut: 11P, 1F, 22M
• Run the cut results.1F-1/2/3 are right. 		• Mix the 1F-1/2 purification products • Cut: 1F-1+1F-2(E+S), 22M-3(X+P), pSB1C3(E+P)
Aug. 24th Wednesday
Aug. 25th Thursday
Aug. 26th Friday
• Amplify: vgb, 1C3
Aug.27th Saturday
• Digest: Vgb, 1C3, fdfhF, 22M+10I, 13K+10I
Aug.28th Sunday
• Ligate: vgb+22M+10I, fdfhF+13K+10I • Transform the ligation results.

 

1st August

Freeze slicing of about 50μm. Observe under natural light microscope and can see red florescence. The thickness is about 130μm

4th August

Repeat the last experiment with silicone tube. 6rpm/s results in a flowing speed of 68ml/day

6th August

Similar to the last time. Did not use freeze slicing.

15th August

Cultured E.coli in 5ml LB for 12h.

16th August

Substituting a part of silicone tube with glass tube in silicone tube biofilm formation sets. Culture in 37℃

17th August

Add 50ml LB and culture with circular culture.

18th August

12.00 found one free-flow pump stopped. Cooled for 15 minutes and turned on again. Could be over heated. Can see some bacteria on the bottom of the vessel and tube. When the pump started again, the bacteria was washed away.

19th August

Can observe obvious white biofilm where the silicone tube joins the tube but cannot see red florescence, suspect contamination. No biofilm is observed on the glass tube under microscope.

21st, August

Biofilm formation with large test tubes. Inoculate with 1% e.coli. Rubber tubes are attached to air pump with filter between air pump and the tube to prevent entrance of germs. Two sets use MSM +glucose medium and two sets use LB.
Retry with glass tube biofilm formation set.

24th, August

Terminate biofilm formation, the glass slide left in the drawer to dry. Can see obvious rod like structure under 400 magnification. (spheres in MSM+glucose culture) No red florescence is seen. Most of the surface is covered by single layer cells but some part of it has thick bump like structure.
No biofilm observed on the glass tube.

Retrieved from "http://2011.igem.org/Team:ZJU-China/Notebook/August"