Difference between revisions of "Team:Munich/assembly2.html"

 
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<h4>T7 and Phage Assembly in commercial cell-extract </h4>
+
<h4>T7 and Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/07/31</em>
 
<em>2018/07/31</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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<tr>
 
<tr>
 
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> T4 Phage: 1,5*10^ 10 PFU/mL
+
       <td> T4 Phage: 4*10^5 PFU/mL
−
T7 Phage: 5*10^9 PFU/mL
+
T7 Phage: 5*10^7 PFU/mL
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
 
   
 
   
−
<h4>T7 Phage Assembly in commercial cell-extract </h4>
+
<h4>T7 Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/08- 2018/08/09</em>
 
<em>2018/08/08- 2018/08/09</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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−
<h4>T4 and 3S Phage Assembly in commercial cell-extract </h4>
+
<h4>T4 and 3S Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/08- 2018/08/09</em>
 
<em>2018/08/08- 2018/08/09</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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<tr>
 
<tr>
 
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
 
       <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
−
       <td> 3S Phage: 2*10^3 PFU/mL
+
       <td> 3S Phage: 2*10^4 PFU/mL
 
T4 Phage: 5*10^4 PFU/mL
 
T4 Phage: 5*10^4 PFU/mL
 
   
 
   
Line 107: Line 107:
 
</table>
 
</table>
 
   
 
   
−
<h4>T7 Phage Burst size Analysis and Assembly in commercial cell-extract </h4>
+
<h4>T7 Phage Burst size Analysis and Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/08</em>
 
<em>2018/08/08</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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</table>
 
</table>
 
   
 
   
−
<h4>T7 Phage Assembly in commercial cell-extract </h4>
+
<h4>T7 Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/10- 2018/08/11</em>
 
<em>2018/08/10- 2018/08/11</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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</table>
 
</table>
 
   
 
   
−
<h4>3S Phage Assembly in commercial cell-extract </h4>
+
<h4>3S Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/07 2018/08/08 </em>
 
<em>2018/08/07 2018/08/08 </em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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−
<h4>T7 Phage Assembly in commercial cell-extract </h4>
+
<h4>T7 Phage Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/13- 2018/08/14</em>
 
<em>2018/08/13- 2018/08/14</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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 +
 
   
 
   
−
<h4>T4 and 3S Phage Assembly in commercial cell-extract </h4>
+
 
−
<em>2018/08/16- 2018/08/17</em>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
      <td>Participants:</td>
+
−
      <td> Brigit Tunaj</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>
+
−
<a href="https://static.igem.org/mediawiki/2018/c/c1/T--Munich--PhageT7AssemblyTXTL.pdf" target="_blank">PhageT7AssemblyTXTL</a>,
+
−
<a href="https://static.igem.org/mediawiki/2018/2/23/T--Munich--AgarOverlayPlaqueAssay_.pdf" target="_blank">Agar overlay plaque assay</a>
+
−
</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td> DNA:300 ng
+
−
    </tr>
+
−
<tr>
+
−
      <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
+
−
      <td> T4 Phage: 1*10^3 PFU/mL
+
−
T7 Phage: 2*10^3 PFU/mL
+
−
</td>
+
−
    </tr>
+
−
+
−
+
−
<h4>T4  Phage Assembly in commercial cell-extract </h4>
+
−
<em>2018/08/21- 2018/08/22</em>
+
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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     </tr>
 
     </tr>
 
   
 
   
−
<h4>T7 Phage Burst size Analysis and Assembly in commercial cell-extract </h4>
+
<h4>T7 Phage Burst Size Analysis and Assembly in Commercial Cell-Extract </h4>
 
<em>2018/08/24-2018/08/25</em>
 
<em>2018/08/24-2018/08/25</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
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</td>
 
</td>
 
     </tr>
 
     </tr>
 +
</table>
 +
 +
<h4>
 +
Burst size with T7
 +
</h4>
 +
<em>
 +
2018/09/21
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2018/0/03/T--Munich--BurstSizeAnalysisHeinemann2007.pdf" target="_blank">Burst size analysis (Heinemann, 2007)</a>,
 +
<a href="https://static.igem.org/mediawiki/2018/2/23/T--Munich--AgarOverlayPlaqueAssay_.pdf" target="_blank">Agar overlay plaque assay </a>
 +
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
Time for adsorption: 5 min instead of 12 min, time until lysis ≈ 1 h
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
Burst size synthetic T7 = 39600 PFU/mL, natural T7 = 9500 PFU/mL
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Amicon MW 100 coprecipitation of T4 phages in cell extract
 +
</h4>
 +
<em>
 +
2018/09/22
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://international.neb.com/protocols/2014/10/24/rapid-pngase-f-by-sds-page-protocol-p0710" target="_blank">SDS-Page</a>
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
No notes.
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
DS Page: no bands (label: P (1 µL T4 + 6.5 µL ddH2O), E ((1 µL T4 + 6.5 µL ddH2O).
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Amicon MW 100 coprecipitation of T4 phages in cell extract
 +
</h4>
 +
<em>
 +
2018/09/24
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://international.neb.com/protocols/2014/10/24/rapid-pngase-f-by-sds-page-protocol-p0710" target="_blank">SDS-Page</a>
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
No notes.
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
Denaturated phage complex in low concentration -> ROTI dye not sensitive enough to show bands specific for phages, significant band at 9 kDa for soc-protein possible.
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Burst size analysis
 +
</h4>
 +
<em>
 +
2018/09/25
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2018/8/83/T--Munich--BurstSizeAnalysis.pdf" target="_blank">Burst size analysis (1:10 Transfection)
 +
</a>
 +
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
No notes.
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
Natural T7 (2,2 x 10^10 PFU/mL) <br>
 +
Synthetic T7 (13.08.2018) (2,36 x 10^11 PFU/mL)
 +
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Plaque Assay Amicon filtered T4 phage
 +
</h4>
 +
<em>
 +
2018/09/26
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="Hhttps://static.igem.org/mediawiki/2018/8/83/T--Munich--BurstSizeAnalysis.pdf" target="_blank">Agar overlay plaque assay </a>
 +
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
Used Amincon filter.
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
2,05 x 10^10 PFU/mL, After: 5,24 x 10^7 PFU/mL.
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Burst size analysis T7 Phage
 +
</h4>
 +
<em>
 +
2018/10/01
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2018/0/03/T--Munich--BurstSizeAnalysisHeinemann2007.pdf" target="_blank">Burst size analysis (Heinemann, 2007)</a>
 +
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
No notes.
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
Ni = Titer 1 - Titer 2 = 5.94 x 10^7 <br>
 +
Burst size = Titer 3/Ni = 202
 +
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h4>
 +
Burst size analysis
 +
</h4>
 +
<em>
 +
2018/10/8
 +
</em>
 +
<table class=”table table-borderless”>
 +
<tr>
 +
<td>Participants:</td>
 +
<td>Sophie Kurzbach</td>
 +
</tr>
 +
<tr>
 +
<td>Protocol:</td>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2018/0/03/T--Munich--BurstSizeAnalysisHeinemann2007.pdf" target="_blank">Burst size analysis (Heinemann, 2007)</a>
 +
 +
</td>
 +
</tr>
 +
<td>Notes:</td>
 +
        <td>
 +
With T7 Phage
 +
      </td>
 +
</tr>
 +
<td>Results:</td>
 +
<td>
 +
burst size: 120
 +
</td>
 +
</tr>
 
</table>
 
</table>
  
 
</div>
 
</div>
 
</html>
 
</html>

Latest revision as of 03:11, 18 October 2018

T7 and Phage Assembly in Commercial Cell-Extract

2018/07/31
Participants: Brigit Tunaj
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: T7 and T4 Phage: 2% PEG, 4% PEG, 6% PEG
Results: T4 4%: 3*10^4 PFU/mL, T7 %, 1*10^8 PFU/mL

Phage Titer from T4 and T7

2018/08/02
Participants: Brigit Tunaj
Protocol: Agar overlay plaque assay
Notes: Standard procedure
Results: T4 Phage: 4*10^5 PFU/mL T7 Phage: 5*10^7 PFU/mL

T7 Phage Assembly in Commercial Cell-Extract

2018/08/08- 2018/08/09
Participants: Sophie Kurzbach
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: DNA from Kilian (10,37 nM)
Results: No plaques

T4 and 3S Phage Assembly in Commercial Cell-Extract

2018/08/08- 2018/08/09
Participants: Brigit Tunaj
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: Standard procedure
Results: 3S Phage: 2*10^4 PFU/mL T4 Phage: 5*10^4 PFU/mL

T7 Phage Burst size Analysis and Assembly in Commercial Cell-Extract

2018/08/08
Participants: Sophie Kurzbach
Protocol: Burst size analysis, PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: No notes
Results: Burst size: 408

T7 Phage Assembly in Commercial Cell-Extract

2018/08/10- 2018/08/11
Participants: Sophie Kurzbach
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: DNA from Kilian (10,37 nM)
Results: No plaques

3S Phage Assembly in Commercial Cell-Extract

2018/08/07 2018/08/08
Participants: Brigit Tunaj
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: Standard procedure
Results: No plaques

T7 Phage Assembly in Commercial Cell-Extract

2018/08/13- 2018/08/14
Participants: Sophie Kurzbach
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: DNA from Kilian
Results: No plaques

T7 Phage Burst Size Analysis and Assembly in Commercial Cell-Extract

2018/08/24-2018/08/25
Participants: Brigit Tunaj
Protocol: PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: Standard procedure
Results: 10^3 PFU/mL
Participants: Sophie Kurzbach
Protocol: Burst size analysis, PhageT7AssemblyTXTL, Agar overlay plaque assay
Notes: Standard procedure
Results: Burst size: 1250

Burst size with T7

2018/09/21
Participants: Sophie Kurzbach
Protocol: Burst size analysis (Heinemann, 2007), Agar overlay plaque assay
Notes: Time for adsorption: 5 min instead of 12 min, time until lysis ≈ 1 h
Results: Burst size synthetic T7 = 39600 PFU/mL, natural T7 = 9500 PFU/mL

Amicon MW 100 coprecipitation of T4 phages in cell extract

2018/09/22
Participants: Sophie Kurzbach
Protocol: SDS-Page
Notes: No notes.
Results: DS Page: no bands (label: P (1 µL T4 + 6.5 µL ddH2O), E ((1 µL T4 + 6.5 µL ddH2O).

Amicon MW 100 coprecipitation of T4 phages in cell extract

2018/09/24
Participants: Sophie Kurzbach
Protocol: SDS-Page
Notes: No notes.
Results: Denaturated phage complex in low concentration -> ROTI dye not sensitive enough to show bands specific for phages, significant band at 9 kDa for soc-protein possible.

Burst size analysis

2018/09/25
Participants: Sophie Kurzbach
Protocol: Burst size analysis (1:10 Transfection)
Notes: No notes.
Results: Natural T7 (2,2 x 10^10 PFU/mL)
Synthetic T7 (13.08.2018) (2,36 x 10^11 PFU/mL)

Plaque Assay Amicon filtered T4 phage

2018/09/26
Participants: Sophie Kurzbach
Protocol: Agar overlay plaque assay
Notes: Used Amincon filter.
Results: 2,05 x 10^10 PFU/mL, After: 5,24 x 10^7 PFU/mL.

Burst size analysis T7 Phage

2018/10/01
Participants: Sophie Kurzbach
Protocol: Burst size analysis (Heinemann, 2007)
Notes: No notes.
Results: Ni = Titer 1 - Titer 2 = 5.94 x 10^7
Burst size = Titer 3/Ni = 202

Burst size analysis

2018/10/8
Participants: Sophie Kurzbach
Protocol: Burst size analysis (Heinemann, 2007)
Notes: With T7 Phage
Results: burst size: 120