Team:Wageningen UR/notebook/journal/kill-switch

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<h1>Journal</h1>
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<h1>Kill-switch journal</h1>
 +
<br/>
<h2>Overview</h2>
<h2>Overview</h2>
<p>
<p>
Primers used to make promoters:
Primers used to make promoters:
 +
<ul>
 +
<li><b>Tet Tet fin primer</b><br/> FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCCTTTCGTCTTCAATAATTCTTGACAAATAACTCTA</li>
 +
<li><b>Tet Tet fin primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAACTCTATCATTGATAGAGTTATTTGTCAAGAATTAT</li>
 +
<li><b>Tet – Tet fin primer</b><br/>  FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCTCCCTATCAGTGATAGAGATTGACATTTATGCTTC</li>
 +
<li><b>Tet – Tet fin primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAATTATACGAGCCGGAAGCATAAATGTCAATCTCTAT</li>
 +
<li><b>CI Lac Lac fin primer</b><br/>  FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTAGACATTGTGAGCGG</li>
 +
<li><b>CI Lac Lac fin Primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGTTGTTACTCGCTCACATTTAAATTGCACGAAGTATCTTGTTATCCGCTCACAATGTCTAACACCGT</li>
 +
<li><b>Lac CI Lac fin primer</b><br/>  FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCAATTGTGAGCGGATAACAATTGACTATTTTACCTC</li>
 +
<li><b>Lac CI Lac fin primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGTTGTTACTCGCTCACATTTAAATTGCACGAATTATCACCGCCAGAGGTAAAATAGTCAATTGTTAT</li>
 +
<li><b>CI Tet Tet fin primer</b><br/>  FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTAGACAAATAACTCTA</li>
 +
<li><b>CI Tet Tet fin primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAACTCTATCATTGATAGAGTTATTTGTCTAACACCGT</li>
 +
<li><b>Tet CI Tet fin primer</b><br/>  FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCTCCCTATCAGTGATAGAGATTGACTATTTTACCTC</li>
 +
<li><b>Tet CI Tet fin primer</b><br/>  RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAATTATCACCGCCAGAGGTAAAATAGTCAATCTCTAT</li>
 +
</ul>
</p>
</p>
<p>
<p>
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<p>
<p>
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The ideas about the kill-switch and what was needed for its construction where finalized. Therefore research was done an suitable parts and solutions where found in papers, the registry and other literature.
+
The ideas about the kill-switch and what was needed for its construction where finalized. Therefore research was done on suitable parts and solutions where found in research papers and the iGEM the registry.
</p>
</p>
<p>
<p>
-
More repressors than the lacI repressor and the tetR repressor where needed so CIλ was used. The promoters that where repressed by combinations of CIλ  and tetR or lacI in the registry where not characterized. Since the ciλ repressor has an important role in the design these promoters had to be characterized. But CIλ is non incunabula so the rhamnose mediated characterization was developed.
+
More repressors than the lacI repressor and the tetR repressor where needed so CIλ was used. The promoters that where repressed by combinations of CIλ  and tetR or lacI in the registry where not characterized. Since the CIλ repressor has an important role in the design these promoters had to be characterized. But CIλ is non inducable so the idea for the rhamnose mediated characterization was developed.
</p>
</p>
<p>
<p>
-
Besides the finalizing of the designs, lab work was prepared by making plates and preparing parts we needed.
+
Besides finalizing the designs, plates were prepared and glycerol stocks were made from needed BioBricks.
</p>
</p>
<p>  
<p>  
-
An <i>in silco</i> assembly of all the characterization plasmids that would be needed and the final system was done. After the <i>in silico</i> experiment flowcharts where made to as an overview of the process.</p>
+
An <i>in silco</i> assembly of all the characterization plasmids and the final system was done.</p>
<p>
<p>
-
You can find the parts we used on the kill-switch page  
+
You can find the used Biobricks on the <a class="soft_link" target=”blank” href=" https://2014.igem.org/Team:Wageningen_UR/project/kill-switch" >Kill-switch</a> page.
</p>
</p>
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<dd class="timelineEvent" id="06w02EX" style="display:none;">
<dd class="timelineEvent" id="06w02EX" style="display:none;">
<p>
<p>
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The promoters from the registry where assembled with GFP and RFP. The assemblies whit GFP where eventually successful but the assembly with RFP did not work.
+
The promoters from the registry where assembled with GFP and RFP. Experiments were continued wirg GFP assembly.
</p>
</p>
<figure>
<figure>
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<img src=" https://static.igem.org/mediawiki/2014/5/57/Wageningen_UR_KS_labnotespic1.png" width="80%">
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<img src="https://static.igem.org/mediawiki/2014/5/57/Wageningen_UR_KS_labnotespic1.png" width="80%">
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<figcaption> Figure 1. </figcaption>
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<figcaption> Figure 1. SYBR-safe stained colony PCR products of promoter/reporter assembly after gel electrophoresis. </figcaption>
</figure><br/>
</figure><br/>
<figure>
<figure>
<img src="https://static.igem.org/mediawiki/2014/2/2c/Wageningen_UR_KS_labnotespic2.png" width="80%">
<img src="https://static.igem.org/mediawiki/2014/2/2c/Wageningen_UR_KS_labnotespic2.png" width="80%">
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<figcaption> Figure 2. </figcaption>
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<figcaption> Figure 2. SYBR-safe stained colony PCR products of promoter/reporter assembly after gel electrophoresis. </figcaption>
</figure><br/><figure>
</figure><br/><figure>
<img src="https://static.igem.org/mediawiki/2014/3/3f/Wageningen_UR_KS_labnotespic3.png" width="80%">
<img src="https://static.igem.org/mediawiki/2014/3/3f/Wageningen_UR_KS_labnotespic3.png" width="80%">
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<p>
<p>
-
The Rhamnose promoter was assembled with the <i>TetR, LacI, CIλ</i> repressor, <i>CIλ</i> combined with <i>TetR</i>, <i>CIλ</i> combined with <i>LacI</i> and GFP so that it could be combined With the different promoters with GFP.
+
The Rhamnose promoter was assembled with the <i>TetR, LacI, CIλ</i> repressors, <i>CIλ</i>+<i>TetR</i>, <i>CIλ</i>+<i>LacI</i> and GFP. Those repressor plasmids were combined with the different promoters+GFP.
</p>
</p>
<p>
<p>
-
The promoters from the registry where also combined with TetR, TetR combined with GFP, lacI and lacI combined with GFP. These parts can be used to construct toggle switches with and without GFP. The toggle switches can be used for validation and characterization of the toggle switch.  
+
The promoters from the registry where also combined with TetR, TetR+GFP, lacI and lacI+GFP. These parts can be used to construct toggle switches with and without GFP. The toggle switches can be used for validation and characterization of the toggle switch.  
</p>
</p>
<figure>
<figure>
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When we performed some test on the two plasmids system and co-transformation we found out that the low copy number plasmid 4A5 had really high miniprep values and didn’t appear to be a low copy number plasmid. </p>
When we performed some test on the two plasmids system and co-transformation we found out that the low copy number plasmid 4A5 had really high miniprep values and didn’t appear to be a low copy number plasmid. </p>
<p>
<p>
-
The model that was made showed that the promoters from the registry with their current operator configuration where not likely to be stable for a long time period. Therefore new promoters had to be created and the necessary research was started.  
+
The <a class="soft_link" href=" https://2014.igem.org/Team:Wageningen_UR/project/model#results2">model</a> that was made showed that the promoters from the registry with their current operator configuration where not likely to be stable for a long time period. Therefore new promoters had to be created and the necessary research was started.  
</p>
</p>
                    
                    
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<h2 class="timelineMajorMarker"><span>October</span></h2>
<h2 class="timelineMajorMarker"><span>October</span></h2>
<dl class="timelineMinor">
<dl class="timelineMinor">
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<dt id="06w04"><a>Week 1-4</a></dt>
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<dt id="06w04"><a>Week 1-2</a></dt>
<dd class="timelineEvent" id="06w04EX" style="display:none;">
<dd class="timelineEvent" id="06w04EX" style="display:none;">
<p>
<p>
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The testing of the protocol was finished and a characterization of the new Ptet promoter (P1) and PcI/Lac from the registry was started.
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The testing of the protocol was finished and a characterization of the new Ptet promoter (P1) and pCI/Lac from the registry was started.
</p>
</p>
<p>
<p>
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</p>
</p>
<p>
<p>
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Products and intermediates where assembled into pSB1C3 and send to iGEM headquarters.
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Products and intermediates where assembled into pSB1C3 and send to iGEM headquarters.</p>
 +
 +
<P>
 +
The following graphs are results from the rhamnose mediated characterization.
</p>
</p>
 +
 +
<br/>
 +
<br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/5/54/Wageningen_UR_characterization_labnotespic1.png" width="80%">
 +
<figcaption> Figure 1. Promoter strength in relative promoter units. RFU values of time point 8.25h are used. A RPU value of pTet (BBa_R0040) 1.5 is used stated as in Kelly et al, 2009. Compared to this known value, pCI/Lac has a RPU of 1.04. </figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/e/e0/Wageningen_UR_characterization_labnotespic2.png" width="80%">
 +
<figcaption> Figure 2. OD600 graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBricks pRha GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. OD is measured in a plate reader over time. </figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/b/b6/Wageningen_UR_characterization_labnotespic3.png" width="80%">
 +
<figcaption> Figure 3. OD600 graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBricks pTet GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. OD is measured in a plate reader over time. </figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d4/Wageningen_UR_characterization_labnotespic4.png" width="80%">
 +
<figcaption> Figure 4. OD600 graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBricks pRha LacI and pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. OD is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/9/96/Wageningen_UR_characterization_labnotespic5.png" width="80%">
 +
<figcaption> Figure 5. OD600 graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBrick pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. OD is measured in a plate reader over time. </figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/5/51/Wageningen_UR_characterization_labnotespic6.png" width="80%">
 +
<figcaption> Figure 6. OD600 graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBricks pRha CIλ and pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. OD is measured in a plate reader over time. </figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/8/8f/Wageningen_UR_characterization_labnotespic7.png" width="80%">
 +
<figcaption> Figure 7. Graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBrick pRha GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. Fluorescence is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/f/f2/Wageningen_UR_characterization_labnotespic8.png" width="80%">
 +
<figcaption> Figure 8. Graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBrick pTet GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. Fluorescence is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/7/73/Wageningen_UR_characterization_labnotespic9.png" width="80%">
 +
<figcaption> Figure 9. Graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBricks pRha LacI and pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. Fluorescence is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/9/95/Wageningen_UR_characterization_labnotespic10.png" width="80%">
 +
<figcaption> Figure 10. Graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBrick pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. Fluorescence is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/e/eb/Wageningen_UR_characterization_labnotespic11.png" width="80%">
 +
<figcaption> Figure 11. Graph of E. coli DH5α containing a pSB3K3 plasmid with the BioBrick pRha CIλ pCI/Lac GFP grown in M9 medium with 2% glycerol and induced at t=0 with different concentrations of rhamnose. Fluorescence is measured in a plate reader over time.</figcaption>
 +
</figure><br/>
 +
 +
 +
                                                     </dd>
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Latest revision as of 03:27, 18 October 2014

Wageningen UR iGEM 2014

 

 

 

Kill-switch journal


Overview

Primers used to make promoters:

  • Tet Tet fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCCTTTCGTCTTCAATAATTCTTGACAAATAACTCTA
  • Tet Tet fin primer
    RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAACTCTATCATTGATAGAGTTATTTGTCAAGAATTAT
  • Tet – Tet fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCTCCCTATCAGTGATAGAGATTGACATTTATGCTTC
  • Tet – Tet fin primer
    RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAATTATACGAGCCGGAAGCATAAATGTCAATCTCTAT
  • CI Lac Lac fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTAGACATTGTGAGCGG
  • CI Lac Lac fin Primer
    RVS:GGCCGCTACTAGTAGTTGGTTGTTACTCGCTCACATTTAAATTGCACGAAGTATCTTGTTATCCGCTCACAATGTCTAACACCGT
  • Lac CI Lac fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCAATTGTGAGCGGATAACAATTGACTATTTTACCTC
  • Lac CI Lac fin primer
    RVS:GGCCGCTACTAGTAGTTGGTTGTTACTCGCTCACATTTAAATTGCACGAATTATCACCGCCAGAGGTAAAATAGTCAATTGTTAT
  • CI Tet Tet fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTAGACAAATAACTCTA
  • CI Tet Tet fin primer
    RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAACTCTATCATTGATAGAGTTATTTGTCTAACACCGT
  • Tet CI Tet fin primer
    FW:GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTACAACGTCGTGTTAGCTGCTCCCTATCAGTGATAGAGATTGACTATTTTACCTC
  • Tet CI Tet fin primer
    RVS:GGCCGCTACTAGTAGTTGGGTAACGCTCTCTATCACTGATAGGGGTGGAATTATCACCGCCAGAGGTAAAATAGTCAATCTCTAT

Protocols used:

Protocols made:

+ expand all


July

Week 1-4

August

Week 1-4

September

Week 1-4

October

Week 1-2



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