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

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<br>
 
<br>
 
We conducted SDS-PAGE with  
 
We conducted SDS-PAGE with  
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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;).
+
(BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on (pSB1C3) of <span style="font-style: italic">E. coli</span> (DH5&alpha;).
 
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.             
 
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.             
−
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.
+
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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   <tr align="center">
 
   <tr align="center">
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     <td>30％ Acrilmid</td>
+
     <td>30% Acrilmid</td>
 
     <td>5 mL</td>
 
     <td>5 mL</td>
 
   </tr>
 
   </tr>
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     <tr align="center">
 
     <tr align="center">
 
     <td>DW</td>
 
     <td>DW</td>
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     <td>2.295 ｍL</td>
+
     <td>2.295 mL</td>
 
   </tr>
 
   </tr>
 
    
 
    
 
     <tr align="center">
 
     <tr align="center">
 
     <td>Total</td>
 
     <td>Total</td>
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     <td>10 ｍL</td>
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     <td>10 mL</td>
 
   </tr>
 
   </tr>
 
    
 
    
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<ol>
 
<ol>
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<li>Centrifuge with 13000rpm at 24&deg;C for 2 min</li>
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<li>Centrifuge with 13000 rpm at 24&deg;C for 2 min</li>
 
<li>Remove the supernatant</li>
 
<li>Remove the supernatant</li>
 
<li>Add 50 mL of SDS-Buffer</li>
 
<li>Add 50 mL of SDS-Buffer</li>

Revision as of 11:40, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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Proof of concept

We conducted SDS-PAGE with (BBa_K2015012 + BBa_K2015008 / BBa_K2015012 + BBa_K2015009) on (pSB1C3) of E. coli (DH5α). 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. BBa_K2015008 consists Self-Assembling Regions (SAR, RADA16-I) and mutated GFP. And BBa_K2015009 This part consists Self-Assembling Region (SAR,P11-4) and mutated GFP. 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.
Table. 1. Gel assay
Materials Volume
30% Acrilmid 5 mL
Tris-Buffer 2.5 mL
SDS 100 µL
APS 100 µL
TEMED 5 µL
DW 2.295 mL
Total 10 mL


The following Table. 2 is about preparing the SDS-PAGE.
Table. 2. Experimental condition
Temperature IPTG Concentration (M) Volume (µL) Time to culture
37°C - - - 24 h
37°C + 0.4 6 24 h
37°C + 2 30 24 h
25°C - - - 16 h
25°C + 0.4 6 16 h
25°C + 2 30 16 h


After we finished cultivating samples, we took 100 µL out of each samples and made the following operation.
  1. Centrifuge with 13000 rpm at 24°C for 2 min
  2. Remove the supernatant
  3. Add 50 mL of SDS-Buffer
  4. Shake for 1 min
  5. Keep at 100°C for 5 min
  6. Put on the ice
  7. Apply 10 µL to SDS
  8. Run electrophoresis for 1.5 h
  9. Wash out with MiliQ
  10. Shake at 24°C with 34 rpm for 1 h
  11. Dye with QuickCBB

The fig. 1 shows the result of SDS-PAGE.