Difference between revisions of "Team:HokkaidoU Japan/Proof"

 
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<br>
 
<br>
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We conducted SDS-PAGE with
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We made a functional unit to regulate gene expression. <a href="http://parts.igem.org/Part:BBa_K2015012">BBa_K2015012</a> codes constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>), RBS (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>), LacI (<a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>), dT (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>), PLac (<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a>) and RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig. 1).
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(BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on (pSB1C3) of <span style="font-style: italic">E.coli</span> (DH5&alpha;).
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Let us explain the 3 parts below. BBa_K2015012 part contains lacI expression unit and plac+RBS. The sequences of downstream can be induced strictly by IPTG.            
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BBa_K2015008 consists Self-Assembling Regions (SAR,RADA16-I) and mutated GFP. And BBa_K2015009 This part consists Self-Assembling Region (SAR,P<span class="sitatuki">11</span>-4) and mutated GFP.
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At first, we made the Gel for SDS-PAGE, with the following the Table. 1. It was necessary to add TEMED finally because the solution was turned into the Gel very fast by TEMED.
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<table style="border-style: none; width:1px; margin-left:auto; margin-right:auto">
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<center>
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<table style="border-style: none">
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<tr align="center">  
 
<tr align="center">  
 
<td style="border-style: none;">  
 
<td style="border-style: none;">  
 
       <tr>
 
       <tr>
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<td style="border-style:none; float:center"><center><span class="small">Table. 1. Gel assay</span></center></td>  
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<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/4/43/T--HokkaidoU_Japan--K2015012-RFP.png" alt="result1" height="400px" width="auto"></td>  
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
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<td style="border-style: none"; align="center">
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<td style="border-style: none"; align="center"><span class="small">Fig. 1. Change of expression by IPTG induction<br>left: not induced, right: induced</span></td>
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<table align="center">
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  <tr align="center">
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<th>Materials</th>
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<th>Volume</th>
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  </tr>
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  <tr align="center">
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    <td>30％ Acrilmid</td>
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    <td>5 mL</td>
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  </tr>
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    <tr align="center">
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    <td>Tris-Buffer</td>
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    <td>2.5 mL</td>
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  </tr>
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    <tr align="center">
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    <td>SDS</td>
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    <td>100 &micro;L</td>
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  </tr>
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    <tr align="center">
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    <td>APS</td>
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    <td>100 &micro;L</td>
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  </tr>
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    <tr align="center">
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    <td>TEMED</td>
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    <td>5 &micro;L</td>
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  </tr>
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    <tr align="center">
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    <td>DW</td>
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    <td>2.295 ｍL</td>
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  </tr>
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    <tr align="center">
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    <td>Total</td>
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    <td>10 ｍL</td>
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  </tr>
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</table>
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</td>
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       </tr>
 
       </tr>
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</tr>
 
</table>
 
</table>
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</center>
 
 
<br clear="all">
 
<br clear="all">
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<br>And also we induced <a href="http://parts.igem.org/Part:BBa_K2015012">BBa_K2015012</a> + <a href="http://parts.igem.org/Part:BBa_K2015008">BBa_K2015008</a> / <a href="http://parts.igem.org/Part:BBa_K2015012">BBa_K2015012</a> + <a href="http://parts.igem.org/Part:BBa_K2015009">BBa_K2015009</a> on pSB1C3 of <span style="font-style: italic">E. coli</span> (DH5&alpha;).           
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<a href="http://parts.igem.org/Part:BBa_K2015008">BBa_K2015008</a> consists Self Assembling Regions (SAR, RADA16-I) and GFP. And <a href="http://parts.igem.org/Part:BBa_K2015009">BBa_K2015009</a> this part consists Self Assembling Region (SAR, P<span class="sitatuki">11</span>-4) and GFP.
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<br>
 
<br>
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  The following Table. 2 is about preparing the SDS-PAGE.  
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<center>
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<br>
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<table style="border-style: none">
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<br>
 +
 
 +
The results are shown the below pictures. The fluorescence of GFPs was not detected any condition.
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</span>
 +
 
 +
<table style="border-style: none; width:1px; margin-left:auto; margin-right:auto">
 
<tr align="center">  
 
<tr align="center">  
 
<td style="border-style: none;">  
 
<td style="border-style: none;">  
 
       <tr>
 
       <tr>
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<td style="border-style:none; float:center"><center><span class="small">Table. 2. Experimental condition</span></center></td>  
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<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/d/da/T--HokkaidoU_Japan--multimerization_BBa_K2015012-BBa_K2015008.png" alt="result1" height="300px" width="auto"></td>  
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
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<td style="border-style: none"; align="center"><span class="small">Fig. 2. BBa_K2015012-BBa_K2015008<br>BBa_K2015012-BBa_K2015008 on pSB1C3 vector were induced by IPTG and incubated at 37&deg;C. (-): not induced, (+): 0.5 mM IPTG and (++): 1.0 mM IPTG</span></td>
 +
      </tr>
 +
</tr>
 +
</table>
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<br clear="all">
  
  
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<table align="center">
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<center>
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  <tr align="center">
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<table style="border-style: none">
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<th>Temperature</th>
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<tr align="center" style="border-style: none">  
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<th>IPTG</th>
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<td style="border-style: none; ">
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<th>Concentration (M)</th>
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<img src="https://static.igem.org/mediawiki/2016/b/be/T--HokkaidoU_Japan--multimerization_BBa_K2015012-BBa_K2015009_25.png" alt="result2" height="300px" width="auto">
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<th>Volume (&micro;L)</th>
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</td>  
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<th>Time to culture</th>
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<td style="border-style: none; ">
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  </tr>
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<img src="https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--multimerization_BBa_K2015012-BBa_K2015009.png" alt="result3" height="300px" width="auto">
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      </td>
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    <tr align="center">
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      </tr>  
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    <td>37&deg;C</td>
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    <td>-</td>
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<td>-</td>
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<td>-</td>
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<td>24 h</td>
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  </tr>
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  <tr align="center">
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    <td>37&deg;C</td>
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    <td>+</td>
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<td>0.4</td>
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<td>6</td>
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<td>24 h</td>
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  </tr>
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    <tr align="center">
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    <td>37&deg;C</td>
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    <td>+</td>
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<td>2</td>
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<td>30</td>
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<td>24 h</td>
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  </tr>
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    <tr align="center">
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    <td>25&deg;C</td>
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    <td>-</td>
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<td>-</td>
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<td>-</td>
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<td>16 h</td>
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−
  </tr>
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    <tr align="center">
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    <td>25&deg;C</td>
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    <td>+</td>
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<td>0.4</td>
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<td>6</td>
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<td>16 h</td>
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−
  </tr>
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−
 
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−
    <tr align="center">
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−
    <td>25&deg;C</td>
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    <td>+</td>
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<td>2</td>
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<td>30</td>
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<td>16 h</td>
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−
  </tr>
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−
 
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</table>
 
</table>
 +
<span class="small">Fig. 3. BBa_K2015012-BBa_K2015009<br>BBa_K2015012-BBa_K2015009 on pSB1C3 vector were induced by IPTG and incubated at 25&deg;C for 24 h (left) <br>and at 37&deg;C for 16 h (right). (-): not induced, (+): 0.5 mM IPTG and (++): 1.0 mM IPTG</span>
 
</center>
 
</center>
 
<br clear="all">
 
<br clear="all">
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</span>
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<br>
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<span class="nomal2">
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After we finished cultivating samples,
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we took 100 &micro;L out of each samples and made the following operation.
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<br>
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<ol>
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<li>Centrifuge with 13000rpm at 24&deg;C for 2 min</li>
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<li>Remove the supernatant</li>
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<li>Add 50 mL of SDS-Buffer</li>
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<li>Shake for 1 min</li>
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<li>Keep at 100&deg;C for 5 min</li>
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<li>Put on the ice</li>
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<li>Apply 10 &micro;L to SDS</li>
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<li>Run electrophoresis for 1.5 h</li>
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<li>Wash out with MiliQ</li>
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<li>Shake at 24&deg;C with 34 rpm for 1 h</li>
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<li>Dye with QuickCBB</li>
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</ol>
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<br>
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The fig. 1 shows the result of SDS-PAGE.
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</span>
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</div>  <!-- 2016contents 閉じる -->
 
</div>  <!-- 2016contents 閉じる -->

Latest revision as of 03:10, 20 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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Proof of concept

We made a functional unit to regulate gene expression. BBa_K2015012 codes constitutive promoter (BBa_J23101), RBS (BBa_B0032), LacI (BBa_C0012), dT (BBa_B0015), PLac (BBa_R0011) and RBS (BBa_B0034). To insert mRFP at the downstream we identified this construct could regulate the expression (Fig. 1).
result1
Fig. 1. Change of expression by IPTG induction
left: not induced, right: induced


And also we induced BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009 on pSB1C3 of E. coli (DH5α). BBa_K2015008 consists Self Assembling Regions (SAR, RADA16-I) and GFP. And BBa_K2015009 this part consists Self Assembling Region (SAR, P11-4) and GFP.


The results are shown the below pictures. The fluorescence of GFPs was not detected any condition.
result1
Fig. 2. BBa_K2015012-BBa_K2015008
BBa_K2015012-BBa_K2015008 on pSB1C3 vector were induced by IPTG and incubated at 37°C. (-): not induced, (+): 0.5 mM IPTG and (++): 1.0 mM IPTG

result2 result3
Fig. 3. BBa_K2015012-BBa_K2015009
BBa_K2015012-BBa_K2015009 on pSB1C3 vector were induced by IPTG and incubated at 25°C for 24 h (left)
and at 37°C for 16 h (right). (-): not induced, (+): 0.5 mM IPTG and (++): 1.0 mM IPTG