Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/Mutation"

 
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<h1 style="margin-bottom: 0px; text-align:left">Lab Notebook</h1>
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<h2 style="color:#ffffff; text-align:left">Documenting the Mutation</h2>
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<div class="container text_header"><h3>Mutation</h3></div><br>
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Firstly, we researched for literature on issues involving <i>in vivo</i> mutagenesis and error prone polymerase. In this phase we read the papers from <a href="http://www.metx.ucsc.edu/research/camps.html
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">Prof. Dr. Manel Camps</a>.
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Discussing the results of the literature research and creating a first draft of what must be done in the laboratory. Further literature research.
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<button data-toggle="collapse" class="btn-block"  data-target="#3">Week 3: (18.04.16-24.04.16): </button>
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<br>
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Looking for suitable parts that can be used for our mutagenesis system. Creating plasmid circuits in geneious. Extension of the first draft.
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Designing of first primers and getting to know the laboratory. Also literature research.
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Contacting the leading expert for the error prone polymerase via e-mail. Dividing the laboratory tasks and creating milestones.
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<button data-toggle="collapse" class="btn-block"  data-target="#6">Week 6: (09.05.16-15.05.16): </button>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;:</li>
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<ul>
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<li>pHSG-WTPolI</li>
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<li>pHSG-EPPolI</li>
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<li>pLA230</li>
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<li>pSBIK3:J04450</li>
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<li>pSBIA3:E0044</li>
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</ul>
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<li>Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&thinsp;&deg;C.</li>
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<li>Plating provided JS200 strain out.</li>
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</ul>
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</div>
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<br>
  
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
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of: </li>
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<li>pHSG-WTPolI</li>
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<li>pHSG-EPPolI</li>
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<li>pLA230</li>
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<li>pSBIK3:J04450</li>
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</ul>
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<li>Overnight cultures from JS200. </li>
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<li>JS200 glycerin stocks (500 &mu;l culture + 200 &mu;l 86% glycerin)</li>
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</ul>
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<br>
  
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Rethinking of our plan.
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<br>
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<ul>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;:</li>
 +
<ul>
 +
<li>pSBIC3:E1010</li>
 +
<li>pSBIC3:K731722</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in JS200;:</li>
 +
<ul>
 +
<li>pHSG-EPPolI & pLA230</li>
 +
<li>pHSG-WTPolI & pLA230</li>
 +
</ul>
 +
<li>Platting out pSBIC3:Isopropanolpathway from iGEM Bielefeld 2014</li>
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</ul>
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</div>
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<br>
  
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<button data-toggle="collapse" class="btn-block"  data-target="#10">Week 10: (06.06.16-12.06.16): </button>
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<li> Overnight cultures of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>  from week 9.</li>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
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</li>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
& <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
with Spe&thinsp;I.</li>
 +
<li>Gel</li>
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<li>Idea: BioBrick site directly behind pMBi ori of pSBIK3</li>
 +
<ul>
 +
<li>1. Deletion of BioBrick site</li>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<ul>
 +
<li>Template: pSBIK3:J04450</li>
 +
<li>Part 1 with primer pSB1K3_Del_for
 +
/pSB1K3_D_for_P2
 +
, temp = 56&thinsp;&deg;C</li>
 +
<li>Part 2 with primer pSB1K3_Del_rev
 +
/pSB1K3_D_rev_P1
 +
, temp = 57&thinsp;&deg;C</li>
 +
<li>-> Part 1 worked; part 2 not</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of pSBIC3:K592100</li>
 +
</ul>
 +
<li>2.: New BioBrick site behind ori</li>
  
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<br>
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<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of K592100</li>
 +
<li>Gradient PCR of part 1 & part 2</li>
 +
</ul>
 +
</div>
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<br>
  
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<ul>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
at 67&thinsp;&deg;C:</li>
 +
<ul>
 +
<li>template: pSBIK3:J04450; primer PS02/PS03</li>
 +
<li>template: pSBIC3:E1010; primer CH30/CH31</li>
 +
<li>template: pSBIC3:K592100; primer PS00/PS01</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a>: 15 &mu;l Gibson Mastermix + 3.5 &mu;l pSBIK3 Backbone + 1.5 &mu;l BFP-Insert</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 +
<li>12 clones on LB-Kan plate that glow blue!</li>
 +
</ul>
 +
</div>
 +
<br>
  
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<li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of the gene synthesis in pSBIC3 backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in NEB electro competent cells</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li>Overnight culture from AraProm, dam-seqA, emrR</li>
 +
</ul>
 +
</div>
 +
<br>
  
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<button data-toggle="collapse" class="btn-block"  data-target="#14">Week 14: (04.07.16-10.07.16): </button>  
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<br>
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<ul>
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iGEM Logo
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
</div>
+
of the overnight cultures</li>
−
<div class="col-md-6 spacer_bottom_10px">
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
−
Irgentwas
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with EcoR&thinsp;I/ Pst&thinsp;I</li>
−
</div>
+
<li>Sequencing of AraProm, dam-seqA, emrR</li>
−
</div>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of pSBIC3:K516132</li>
 +
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
 +
<li>overnight culture of pSBIC3:K516132</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of pSBIC3:K516132</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
with template pSBIC3:K516132, primer CH30/CH31, temp = 67 &deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
of pcr product pSBIC3:K516132, 1h 37&thinsp;&deg;C</li>
 +
<li>1% Agarosegel & purification of the 2&thinsp;kb band -> new backbone</li>
 +
<li>repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> with the new backbone and ugi-cda1/ dnaQ</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 +
<ul>
 +
<li>pSBIA3:J04450</li>
 +
<li>pSBIC3:J06702</li>
 +
<li>GFP-cd</li>
 +
<li>pSBIC:E2050</li>
 +
<li>dnaQ Gibson</li>
 +
<li>ugi-cda1 Gibson</li>
 +
</ul>
 +
<li>Overnight culture of DH5&alpha; with pSBIK3:RFP-cd/ pSBIK3:BFP-cd </li>
 +
<li>Testing of the fluorescent proteins RFP and BFP with the Teacan</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIC3:dnaQ with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li>
 +
</ul>
 +
</div>
 +
<br>
  
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<button data-toggle="collapse" class="btn-block" data-target="#15">Week 15: (11.07.16-17.07.16): </button>  
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<ul>
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+
<li>Primer design for genesynthesis</li>
−
<li data-target="#myCarousel" data-slide-to="2"></li>
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
−
</ol>
+
of the overnight cultures</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of pSBIC3:dnaQ, mCherry, E2050-cds</li>
 +
<li>Sequencing of dnaQ clones with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with CH34/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
on ugi-cda1</li>
 +
<li>Sequencing of ugi-cda1</li>
 +
<li>Assembly of dnaQ, dam, seqA</li>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<ul>
 +
<li>template pSBIC3:dnaQ, primer dnaQ_Gib_for
 +
/dnaQ_Gib_rev
 +
, temp = 59.9&thinsp;&deg;C</li>
 +
<li>template pSBIC3:dam-seqA, primer dam_Gib_for
 +
/dam_Gib_rev
 +
, temp = 58.3&thinsp;&deg;C</li>
 +
<li>template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57&thinsp;&deg;</li>
 +
<li>template pHSG-WTPolI, primer PolI_Gib_for
 +
/PolI_Gib_rev
 +
, temp = 58.4&thinsp;&deg;C</li>
 +
<li>template pHSG-EPPolI, primer PolI_Gib_for
 +
/PolI_Gib_rev
 +
, temp = 58.4&thinsp;&deg;C</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
, <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of pSBIC3:RFP-cd with Xba&thinsp;I/Spe&thinsp;I, <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a>
 +
of 2&thinsp;kb band</li>
 +
</ul>
 +
</ul>
 +
</div>
 +
<br>
  
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<button data-toggle="collapse" class="btn-block"  data-target="#16">Week 16: (18.07.16-24.07.16): </button>  
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<div id="16" class="collapse">
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<div class="item active">
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<br>
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<img src="http://www.mittelstand-die-macher.de/media/cache/article_content/cms/2016/04/Kaufverhalten-Farben.jpg" alt="A">
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<ul>
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</div>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA, C3 backbone</li>
−
<div class="item">
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX compis</li>
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<img src="http://www.lonecke-zetel.de/uploads/pics/Farben_19.jpg" alt="B">
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
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</div>
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with the Gibson clones -> no bands</li>
−
<div class="item">
+
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
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<img src="http://de.wallpaperhd.biz/wp-content/uploads/2013/01/hd-wallpaper-farben-800x600.jpg" alt="C">
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</li>
−
</div>
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<ul>
−
</div>
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<li>template pSBIC3:dnaQ, primer CH35/36, temp = 59.9&thinsp;&deg;C</li>
 +
<li> template pSBIC3:dam-seqA, primer CH37/38, temp = 58.3&thinsp;&deg;C </li>
 +
<li> template pSBIC3:dam-seqA, primer CH39/40, temp = 57&thinsp;&deg;C </li>
 +
<li> template pSBIK3:J04450, primer CH30/31, temp = 67&thinsp;&deg;C </li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a>
 +
of pSBIK3 backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA in K3 backbone (ASI)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 +
</ul>
 +
</div>
 +
<br>
  
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<!-- Left and right controls -->
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<button data-toggle="collapse" class="btn-block" data-target="#17">Week 17: (25.07.16-31.07.16): </button>
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<span class="sr-only">Previous</span>
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</a>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
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<a class="right carousel-control" href="#myCarousel" role="button" data-slide="next">
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of ASI with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
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<span class="glyphicon glyphicon-chevron-right" aria-hidden="true"></span>
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/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> -> plating positive clones out</li>
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<span class="sr-only">Next</span>
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<li>Sequencing of positive clones</li>
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</a>
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
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</div>
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</li>
 +
<ul>
 +
<li>template pSBIC3:emrR, primer BF1/2</li>
 +
<li> template pSBIC3:ugi-cda1, primer BF4/5</li>
 +
<li> template pSBIC3:emrR, primer BF6/7</li>
 +
<li> template pHSG:EPPolI, primer CH42/43</li>
 +
<li> template pHSG:WTPolI, primer CH42/43</li>
 +
</ul>
 +
<li>PCR clean up of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of EPPolI/ WTPolI in pSBIK3 backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 +
</ul>
 +
</div>
 +
<br>
  
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<button data-toggle="collapse" class="btn-block" data-target="#18">Week 18: (01.08.16-07.08.16): </button>  
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<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>  
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<li class="navbar_button"><a href="#">TEAM</a>
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-> ASII did not work</li>
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<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">colony PCR</a>  
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<a href="#">Official Team Profile</a>
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-> sequencing of positive clones</li>
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                                                <a href="#"> Collaborations</a>
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                                                    <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Team/Collaborations/Postcards"> Postcards </a></li>
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                                                <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Notebook"> Notebook</a>
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                                                    <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Notebook/Library"> Library </a></li>
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                                                    <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Notebook/Mutation"> Mutation </a></li>
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                                                    <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Notebook/Selection"> Selection </a></li>
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                                                </ul>
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<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Library">Library</a>
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<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Mutation">Mutation</a>
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−
<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Selection">Selection</a>
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</div>
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</li>
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<li class="navbar_button"><a href="#">PARTS</a></li>
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<li class="navbar_button"><a href="#">SAFETY</a></li>
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<li class="navbar_button"><a href="#">ATTRIBUTIONS</a></li>
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<li class="navbar_button"><a href="#">HUMAN PRACTICES</a></li>
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        <div class="maincontentOfpage">
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            <h1>Lab Notebook Mutation</h1>
+
  
−
        </div>
+
<button data-toggle="collapse" class="btn-block"  data-target="#19">Week 19: (08.08.14-14.08.16): </button>
 +
<div id="19" class="collapse">
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K584001 & K608010</li>
 +
<li>Planning of reversion experiments</li>
 +
</ul>
 +
</div>
 +
<br>
  
 +
<button data-toggle="collapse" class="btn-block"  data-target="#20">Week 20: (15.08.16-21.08.16): </button>
 +
<div id="20" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Sequencing results: mutagenesis ASI has mutations, ASII did not work</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of ASII Gibson</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of ASI/ ASII clones</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of ASII</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of positive clones of ASII</li>
 +
<li>Repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">colony PCR</a>
 +
of ASI with primer cPCR_dnaQ_fw/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
-> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of right ASII clones with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of right ASI clone with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
</ul>
 
</div>
 
</div>
−
<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.12.4/jquery.min.js"></script>
+
<br>
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<script>window.jQuery || document.write('<script src="../../assets/js/vendor/jquery.min.js"><\/script>')</script>
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<script src="http://getbootstrap.com/dist/js/bootstrap.min.js"></script>
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<button data-toggle="collapse" class="btn-block"  data-target="#21">Week 21: (22.08.16-28.08.16): </button>
 +
<div id="21" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">PCR</a> for coloning ASI into pSBIC3</li>
 +
<ul>
 +
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6&thinsp;&deg;C</li>
 +
<li>template pSBIC3:RFP-cd, primer CH30/31, temp = 67&thinsp;&deg;C</li>
 +
<li>template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6&thinsp;&deg;C</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
<ul>
 +
<li>pSBIK3:J04450</li>
 +
<li> pSBIC3:K608010</li>
 +
<li> pSBIC3:K584001</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of positive emrR clones and positive ASII clone, strong RFP</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of ASII and strong RFP</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> into DH5&alpha;</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of pSBIK3, K608010, K584001</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> of pSBIK3 + K608010</li>
 +
<li>Overnight culture of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of PCR products & PCR clean up</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 +
<ul>
 +
<li>pSBIC3:AraC-Pbad (non-leaky)</li>
 +
<li>C3 backbone + ASI clone 4</li>
 +
<li>C3 backbone + ASI clone 15</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> & <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha;</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
</li>
 +
<ul>
 +
<li>template pSBIC3:AraCPbad-nonleaky, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/CH53, temp = 58&thinsp;&deg;C</li>
 +
<li>template pSBIC3:ASI, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/VF_rev, temp = 58&thinsp;&deg;C </li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<ul>
 +
<li> template A68T_A, primer CH20/A68T_rev, temp = 58.6&thinsp;&deg;C (AA)</li>
 +
<li> template A68T_B, primer A68T_B/CH21, temp = 58.5&thinsp;&deg;C (AB)</li>
 +
<li> template C379A_A, primer CH20/C379A_rev, temp = 57.7&thinsp;&deg;C (BA)</li>
 +
<li> template C379A_B, primer C379A_fw/CH21, temp = 58&thinsp;&deg;C (BB)</li>
 +
<li> template A380T_A, primer CH20/ A380T_rev, temp = 58.1&thinsp;&deg;C (CA)</li>
 +
<li> template A380T_B, primer A380T_fw/CH21, temp = 58.1&thinsp;&deg;C (CB)</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX</li>
 +
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of AraC-Pbad (non leaky), ASI</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5&thinsp;&deg;C</li>
 +
<li>Sequencing of pSBIC3:emrR-1/-2 with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIK3:K608010_A/B/C with CH50/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
, temp = 50&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of C3 backbone with EcoR&thinsp;I/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K516031 in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of pSBIC3:K608010_StopA/B/C with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
<li>Sequencing A with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, B with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
and C with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#22">Week 22: (29.08.16-04.09.16): </button>
 +
<div id="22" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
of Stop-GFPs -> Sequencing</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
</li>
 +
<ul>
 +
<li>pSBIC3:AraCPbad with Spe&thinsp;I, Pst&thinsp;I</li>
 +
<li> pSBIC3:AraCPbad(non-leaky) with Spe&thinsp;I, Pst&thinsp;I </li>
 +
<li> pSBIC3:RFP Generator with Xba&thinsp;I, Pst&thinsp;I </li>
 +
<li> pSBIK3:MP6-I-Klon 4 with Spe&thinsp;I, Pst&thinsp;I </li>
 +
<li> pSBIK3:MP6-II-Klon with Xba&thinsp;I, Pst&thinsp;I </li>
 +
</ul>
 +
<li>Designing of new primer</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> </li>
 +
<ul>
 +
<li>C3:AraC-Pbad + RFP-Generator</li>
 +
<li>C3:AraC-Pbad(non-leaky) + RFP-Generator</li>
 +
<li>K3:Mut-I + Mut II</li>
 +
<li>C3 + MutII</li>
 +
</ul>
 +
<li>Repeat of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">restriction digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a>, pHSG-EPPolI, pHSG-WTPolI in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with V<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>
 +
of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII</li>
 +
<li>Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 57&thinsp;&deg;C</li>
 +
<ul>
 +
<li>template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61</li>
 +
<li> template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57</li>
 +
<li> template C3:dnaQ, primer CH54/CH55</li>
 +
<li> template pHSG-WTPolI, primer CH58/C59</li>
 +
<li> template pHSG-WTPolI, primer CH42/C43</li>
 +
<li> template pHSG-EPPolI, primer CH58/C59</li>
 +
<li> template pHSG-EPPolI, primer CH42/C43</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li>
 +
<ul>
 +
<li>damseqA + C3</li>
 +
<li>ugi-cda1 + C3</li>
 +
<li>WTPolI-Part + C3</li>
 +
<li>EPPolI-Part + C3</li>
 +
<li>dnaQ-Insert + dnaQ-BB</li>
 +
<li>WTPolI-Expression + PolI-BB</li>
 +
<li>EPPolI-Expression + PolI-BB</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, temp = 57&thinsp;&deg;C</li>
 +
<ul>
 +
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li>
 +
<li>C3:ugi-cda1</li>
 +
<li>C3:AraC-Pbad(tight)-B0031-dnaQ</li>
 +
<li>C3:EPPolI</li>
 +
<li>C3:AraC-Pbad(tight)-B0031-EPPolI</li>
 +
<li>C3:seqA</li>
 +
<li>C3:WTPolI</li>
 +
</ul>
 +
<li>Sequencing</li>
 +
<li>Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:AraC-Pbad(tight)-B0031-E1010 in Top10</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#23">Week 23: (05.09.16-11.09.16): </button>
 +
<div id="23" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of ugi-cda1 -> Sequencing of right clones</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 58&thinsp;&deg;C</li>
 +
<ul>
 +
<li>template C3:damseqA, primer CH71/72</li>
 +
<li>template C3:StoppGFP Stop A Stop B, primer CH20/66</li>
 +
<li>template C3:damseqA, primer CH73/74</li>
 +
<li>template K3:Stopp GFP A+B, primer CH64/21</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of Stop A, Stop B</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> stop A + stop B</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
with EcoR&thinsp;I/Pst&thinsp;I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:PRha, C3:PRha-GFP-Generator in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of PRha-GFP-Generator in top10</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of StoppA+B in DH5&alpha;</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#24">Week 24: (12.09.16-18.09.16): </button>
 +
<div id="24" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
, temp = 58&deg;C</li>
 +
<ul>
 +
<li>template C3:dnaQ, primer 54/55</li>
 +
<li> template C3:WTPolI (CWP1), primer 58/59</li>
 +
<li> template C3:EPPolI (CEP1), primer 58/59</li>
 +
<li> template C3:ASI+ASII, primer 54/76</li>
 +
<li> template C3:K516031, primer 56/57</li>
 +
<li> template C3:K516031, primer 60/61</li>
 +
<li> template C3:K516031, primer 60/61</li>
 +
<li> template C3:K516031, primer 75/57</li>
 +
</ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of seqA</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5&alpha; of C3:seqA, K3:StopA+B</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
of C3:damseqA with CH71/72, temp = 58&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> in DH5&alpha;</li>
 +
<li>Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 +
<li>Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of dnaQ-Gen, MP6-Gen with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> and CH77/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
</li>
 +
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
of C3:damseqA, primer 71/72, gradient 55-59&thinsp;&deg;C/li>
 +
<li>Sequencing of stop A+B</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of M6 in RFPGen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li>
 +
<li>Test cultivation of a 96-well plate</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of M6-Gen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Plasmid isolation</a>
 +
</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of dnaQ-Gen with Xba&thinsp;I/Pst&thinsp;I</li>
 +
<li>araE-PCR with temp 58&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of araE</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">PCR</a> of DNA, primer 81/82 and C3:med RFP 83/84</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a>
 +
of dnaQ-Gen</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> with C3:AraC-Pbad/C3:PRha</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a>
 +
</li>
 +
<li>PCR clean up</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of araE-Insert + araE-backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> in KRX</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of C3:araE-Device</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of C3 backbone + ugi_cda1, C3 backbone + ASII</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly in DH5&alpha;</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>, Prha-B0031-dnaQ-Ter with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#25">Week 25: (19.09.16-25.09.16): </button>
 +
<div id="25" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of M6-Generator with Xba&thinsp;I/Pst&thinsp;I, C3:K808000, C3:Prha with Spe&thinsp;I/Pst&thinsp;I</li>
 +
<li>Clean up</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Ligation</a> with backbone</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a>, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&thinsp;&deg;C</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:AraC(tight)dnaQ + plA230 in Top10</li>
 +
<li>Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ</li>
 +
<li>Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ</li>
 +
<li>Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2&thinsp;h growth, +25mM arabinose/25mM glycose</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of JS200 EP/WT with plA230</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a>
 +
of AraC-Pbad-M6; Prha-M6</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 in JS00 EP/WT</li>
 +
<li>Rifampicillin assay</li>
 +
<li>beta-lactamase Reversions assay</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of plA230 + C3:Pbad(med)-dnaQ in Top10</li>
 +
<li>Overnight cultures</li>
 +
</ul>
 +
<br>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#26">Week 26: (26.09.16-02.10.16):</button>
 +
<div id="26" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture</li>
 +
<li>Isolation of JS200 + PolI+plA230</li>
 +
<li>Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)</li>
 +
<li>Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6</li>
 +
<li>Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 +
<li>Reversions assay</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a>
 +
of E/P of pSB4C5(E/P)</li>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a>
 +
</li>
 +
<ul>
 +
<li>template C3:ugi-cda, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>
 +
, temp = 58&thinsp;&deg;C</li>
 +
<li>template C3:cda, primer 69/70, temp = 60&thinsp;&deg;C</li>
 +
<li>template A3, primer CK06/05, temp = 59.9&thinsp;&deg;C</li>
 +
<li>template reporter, primer CK07/08, temp = 59&thinsp;&deg;C</li>
 +
 
 +
</ul>
 +
</ul>
 +
<br>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#27">Week 27: (03.10.16-09.10.16): </button>
 +
<div id="27" class="collapse">
 +
<br>
 +
<ul>
 +
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10</li>
 +
<li>Reversions assay</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" class="btn-block"  data-target="#28">Week 28: (10.10.16-16.10.16): </button>
 +
<div id="28" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Analysis reversion assays</li>
 +
<li>Analysis of MiSeq data</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<br>
 +
 
 +
 +
 
 +
 
 +
 
 +
</div>
 +
 
 +
 
 +
 
 +
</div>
 +
 
 +
<script type="text/javascript" src="https://2016.igem.org/wiki/index.php?title=Template:Team:Bielefeld-CeBiTec/styles/js&amp;action=raw&amp;ctype=text/javascript"></script>
 +
</body>
 
</html>
 
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Latest revision as of 16:11, 1 December 2016



Lab Notebook

Documenting the Mutation

Mutation



Firstly, we researched for literature on issues involving in vivo mutagenesis and error prone polymerase. In this phase we read the papers from Prof. Dr. Manel Camps.


Discussing the results of the literature research and creating a first draft of what must be done in the laboratory. Further literature research.


Looking for suitable parts that can be used for our mutagenesis system. Creating plasmid circuits in geneious. Extension of the first draft.


Designing of first primers and getting to know the laboratory. Also literature research.


Contacting the leading expert for the error prone polymerase via e-mail. Dividing the laboratory tasks and creating milestones.


  • Transformation in DH5α:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • pSBIK3:J04450
    • pSBIA3:E0044
  • Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37 °C.
  • Plating provided JS200 strain out.


  • Plasmid isolation of:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • pSBIK3:J04450
  • Overnight cultures from JS200.
  • JS200 glycerin stocks (500 μl culture + 200 μl 86% glycerin)


Rethinking of our plan.


  • Transformation in DH5α:
    • pSBIC3:E1010
    • pSBIC3:K731722
  • Transformation in JS200;:
    • pHSG-EPPolI & pLA230
    • pHSG-WTPolI & pLA230
  • Platting out pSBIC3:Isopropanolpathway from iGEM Bielefeld 2014


  • Overnight cultures of transformation from week 9.
  • Plasmid isolation
  • Plasmid isolation & restriction digestion with Spe I.
  • Gel
  • Idea: BioBrick site directly behind pMBi ori of pSBIK3
    • 1. Deletion of BioBrick site
      • Q5 PCR
        • Template: pSBIK3:J04450
        • Part 1 with primer pSB1K3_Del_for /pSB1K3_D_for_P2 , temp = 56 °C
        • Part 2 with primer pSB1K3_Del_rev /pSB1K3_D_rev_P1 , temp = 57 °C
        • -> Part 1 worked; part 2 not
      • Transformation in DH5α of pSBIC3:K592100
    • 2.: New BioBrick site behind ori




  • Q5 PCR at 67 °C:
    • template: pSBIK3:J04450; primer PS02/PS03
    • template: pSBIC3:E1010; primer CH30/CH31
    • template: pSBIC3:K592100; primer PS00/PS01
  • Gibson assembly: 15 μl Gibson Mastermix + 3.5 μl pSBIK3 Backbone + 1.5 μl BFP-Insert
  • Transformation in DH5α
  • 12 clones on LB-Kan plate that glow blue!


  • Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer
  • Gibson assembly of the gene synthesis in pSBIC3 backbone
  • Transformation in NEB electro competent cells
  • Colony PCR with VR/VF
  • Overnight culture from AraProm, dam-seqA, emrR


  • Plasmid isolation of the overnight cultures
  • Restriction digestion with EcoR I/ Pst I
  • Sequencing of AraProm, dam-seqA, emrR
  • Transformation in DH5α of pSBIC3:K516132
  • Repeated Colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer
  • overnight culture of pSBIC3:K516132
  • Plasmid isolation of pSBIC3:K516132
  • Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, temp = 67 °C
  • DPN1 digestion of pcr product pSBIC3:K516132, 1h 37 °C
  • 1% Agarosegel & purification of the 2 kb band -> new backbone
  • repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ
  • Transformation in DH5α
    • pSBIA3:J04450
    • pSBIC3:J06702
    • GFP-cd
    • pSBIC:E2050
    • dnaQ Gibson
    • ugi-cda1 Gibson
  • Overnight culture of DH5α with pSBIK3:RFP-cd/ pSBIK3:BFP-cd
  • Testing of the fluorescent proteins RFP and BFP with the Teacan
  • Colony PCR of pSBIC3:dnaQ with VR/VF
  • Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702


  • Primer design for genesynthesis
  • Plasmid isolation of the overnight cultures
  • Restriction digestion of pSBIC3:dnaQ, mCherry, E2050-cds
  • Sequencing of dnaQ clones with VR /VF
  • Colony PCR with CH34/VR on ugi-cda1
  • Sequencing of ugi-cda1
  • Assembly of dnaQ, dam, seqA
    • Q5 PCR
      • template pSBIC3:dnaQ, primer dnaQ_Gib_for /dnaQ_Gib_rev , temp = 59.9 °C
      • template pSBIC3:dam-seqA, primer dam_Gib_for /dam_Gib_rev , temp = 58.3 °C
      • template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57 °
      • template pHSG-WTPolI, primer PolI_Gib_for /PolI_Gib_rev , temp = 58.4 °C
      • template pHSG-EPPolI, primer PolI_Gib_for /PolI_Gib_rev , temp = 58.4 °C
    • DPN1 digestion , Gel extraction
    • Restriction digestion of pSBIC3:RFP-cd with Xba I/Spe I, gel extraction of 2 kb band




  • Colony PCR of ASI with VR /VF -> plating positive clones out
  • Sequencing of positive clones
  • Q5 PCR
    • template pSBIC3:emrR, primer BF1/2
    • template pSBIC3:ugi-cda1, primer BF4/5
    • template pSBIC3:emrR, primer BF6/7
    • template pHSG:EPPolI, primer CH42/43
    • template pHSG:WTPolI, primer CH42/43
  • PCR clean up of the Q5 PCR
  • Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)
  • Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone
  • Transformation in DH5α




  • Transformation of pSBIC3:K584001 & K608010
  • Planning of reversion experiments




  • Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)
  • PCR for coloning ASI into pSBIC3
    • template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6 °C
    • template pSBIC3:RFP-cd, primer CH30/31, temp = 67 °C
    • template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6 °C
  • DPN1 digestion
  • Restriction digestion with EcoR I/Pst I
    • pSBIK3:J04450
    • pSBIC3:K608010
    • pSBIC3:K584001
  • Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
  • Restriction digestion with EcoR I/Pst I of ASII and pSBIC3:Strong RFP
  • Ligation of ASII and strong RFP
  • Transformation of the ligation into DH5α
  • Gel extraction of pSBIK3, K608010, K584001
  • Ligation of pSBIK3 + K608010
  • Overnight culture of the ligation
  • Gel extraction of PCR products & PCR clean up
  • Gibson assembly
    • pSBIC3:AraC-Pbad (non-leaky)
    • C3 backbone + ASI clone 4
    • C3 backbone + ASI clone 15
  • Ligation & Transformation in DH5α
  • Colony PCR
    • template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58 °C
    • template pSBIC3:ASI, primer VR /VF_rev, temp = 58 °C
  • Q5 PCR
    • template A68T_A, primer CH20/A68T_rev, temp = 58.6 °C (AA)
    • template A68T_B, primer A68T_B/CH21, temp = 58.5 °C (AB)
    • template C379A_A, primer CH20/C379A_rev, temp = 57.7 °C (BA)
    • template C379A_B, primer C379A_fw/CH21, temp = 58 °C (BB)
    • template A380T_A, primer CH20/ A380T_rev, temp = 58.1 °C (CA)
    • template A380T_B, primer A380T_fw/CH21, temp = 58.1 °C (CB)
  • Gel extraction
  • Gibson assembly
  • Transformation in KRX
  • Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64
  • Colony PCR of AraC-Pbad (non leaky), ASI
  • Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C
  • Sequencing of pSBIC3:emrR-1/-2 with VR /VF
  • Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR , temp = 50 °C
  • Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010
  • Restriction digestion of C3 backbone with EcoR I/Pst I
  • Transformation of pSBIC3:K516031 in KRX
  • Colony PCR of pSBIC3:K608010_StopA/B/C with VR /VF
  • Sequencing A with VF, B with VR and C with VR


  • Plasmid isolation of Stop-GFPs -> Sequencing
  • Restriction digestion
    • pSBIC3:AraCPbad with Spe I, Pst I
    • pSBIC3:AraCPbad(non-leaky) with Spe I, Pst I
    • pSBIC3:RFP Generator with Xba I, Pst I
    • pSBIK3:MP6-I-Klon 4 with Spe I, Pst I
    • pSBIK3:MP6-II-Klon with Xba I, Pst I
  • Designing of new primer
  • Gel extraction of the restriction digestion
  • Ligation
    • C3:AraC-Pbad + RFP-Generator
    • C3:AraC-Pbad(non-leaky) + RFP-Generator
    • K3:Mut-I + Mut II
    • C3 + MutII
  • Repeat of restriction digestion of ASII: template K3:ASII, EcoR I-Hf/Pst I
  • Gel extraction of the restriction digestion
  • Ligation
  • Transformation of the ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
  • Colony PCR with VVR /VF of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII
  • Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose
  • Q5 PCR , temp = 57 °C
    • template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61
    • template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57
    • template C3:dnaQ, primer CH54/CH55
    • template pHSG-WTPolI, primer CH58/C59
    • template pHSG-WTPolI, primer CH42/C43
    • template pHSG-EPPolI, primer CH58/C59
    • template pHSG-EPPolI, primer CH42/C43
  • DPN1 digestion
  • Gel extraction
  • Gibson assembly
    • damseqA + C3
    • ugi-cda1 + C3
    • WTPolI-Part + C3
    • EPPolI-Part + C3
    • dnaQ-Insert + dnaQ-BB
    • WTPolI-Expression + PolI-BB
    • EPPolI-Expression + PolI-BB
  • Transformation
  • Colony PCR with VR /VF, temp = 57 °C
    • C3:AraC-Pbad(tight)-B0031-WTPolI
    • C3:ugi-cda1
    • C3:AraC-Pbad(tight)-B0031-dnaQ
    • C3:EPPolI
    • C3:AraC-Pbad(tight)-B0031-EPPolI
    • C3:seqA
    • C3:WTPolI
  • Sequencing
  • Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10
  • Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10






  • Restriction digestion of M6-Generator with Xba I/Pst I, C3:K808000, C3:Prha with Spe I/Pst I
  • Clean up
  • Ligation with backbone
  • Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI
  • Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37 °C
  • Transformation of C3:AraC(tight)dnaQ + plA230 in Top10
  • Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ
  • Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ
  • Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2 h growth, +25mM arabinose/25mM glycose
  • Transformation of JS200 EP/WT with plA230
  • Colony PCR of AraC-Pbad-M6; Prha-M6
  • Transformation of plA230 in JS00 EP/WT
  • Rifampicillin assay
  • beta-lactamase Reversions assay
  • Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10
  • Overnight cultures



  • Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10
  • Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture
  • Isolation of JS200 + PolI+plA230
  • Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)
  • Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation
  • Restriction digestion of the MiSeq probes: WT, EP, pLA230, pLA230+dnaQ; pLA230+M6
  • Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)
  • Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
  • Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
  • Reversions assay
  • Restriction digestion of E/P of pSB4C5(E/P)
  • Q5 PCR
    • template C3:ugi-cda, primer VF/VR , temp = 58 °C
    • template C3:cda, primer 69/70, temp = 60 °C
    • template A3, primer CK06/05, temp = 59.9 °C
    • template reporter, primer CK07/08, temp = 59 °C



  • Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10
  • Reversions assay


  • Analysis reversion assays
  • Analysis of MiSeq data