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

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<div class="container text_header"><h1>lab notebook.</h1></div>
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<div class="container text_header"><h3>Mutation</h3></div>
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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 paper from [Manel Camps].
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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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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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Transformation in DH5&alpha;:
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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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Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&deg;C.
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Platting provided JS200 strain out.
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<li>Plasmid isolation of: </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>Overnight cultures from JS200. </li>
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<li>JS200 glycerin stocks (500 &mu; culture + 200 &mu; 86% glycerin)</li>
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Rethinking of our plan.
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<li>Transformation in DH5&alpha;:</li>
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<li>psBIC3:E1010</li>
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<li>psBIC3:K731722</li>
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<li>Transformation in JS200;:</li>
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<li>pHSG-EPPolI & plA230</li>
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<li>pHSG-WTPolI & plA230</li>
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</ul>
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<li>Platting out pSBIC3:Isopropanolpathway from iGEM Bielefeld 2014</li>
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<li> Overnight cultures of transformation from week 9.</li>
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<li>Plasmid isolation & restriction with SpeI. </li>
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<li>Idea: BioBrick site directli behind pMBi ori of psBIK3</li>
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<li>1. Deletion of BioBrick site</li>
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<li>Q5 PCR</li>
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<li>Template: pSBIK3:J04450</li>
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<li>part 1 with primer CH07/CH21, tm=56&deg;C</li>
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<li>part 2 with primer CH08/CH20, tm=57&deg;C</li>
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<li>-> part 1 worked; part 2 not</li>
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</ul>
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<li>Transformation in DH5&alpha; of pSBIC3:K592100</li>
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</ul>
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<li>2.: New BioBrick site behind ori</li>
  
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<br>
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<ul>
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<li>Restriction of pSBIK3:J04450 with SPEI and Xba</li>
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<li>colony PCR of K592100</li>
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<li>gradient PCR of part 1 & part 2</li>
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<li>Q5 PCR at 67&deg;C:</li>
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<ul>
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<li>template: PSBIK3:J04450; primer PS02/PS03</li>
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<li>template: PSBIC3:E1010; primer CH30/CH31</li>
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<li>template: PSBIC3:K592100; primer PS00/PS01</li>
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</ul>
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<li>Gibson-Assembly: 15 &mu; Gibson Mastermix + 3.5 &mu; pSBIK3 Backbone + 1.5 &mu; BFP-Insert</li>
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<li>Transformation in DH5&alpha;</li>
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<li>12 clones on LB-Kan plate that glow blue!</li>
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</ul>
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</div>
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<br>
  
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<br>
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<ul>
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<li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li>
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<li>Gibson assembly of the gene synthesis in pSBIC3 backbone</li>
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<li>Transformation in NEB electro competent cells</li>
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<li>Colony PCR with VR/VF</li>
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<li>Overnight culture from AraProm, dam-seqA, emrR</li>
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</ul>
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</div>
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<br>
  
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<li>Plasmid isolation of the overnight cultures</li>
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<li>Restriction with EcoRI/ PstI</li>
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<li>Sequencing of AraProm, dam-SeqA, emrR</li>
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<li>Transformation in DH5&alpha; of pSBIC3:K516132</li>
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<li>Repeated colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
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<li>overnight culture of pSBIC3:K516132</li>
 +
<li>Plasmid isolation of pSBIC3:K516132</li>
 +
<li>Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, tm = 67 &deg;C</li>
 +
<li>DpnI digestion of pcr product pSBIC3:K516132, 1h 37&deg;C</li>
 +
<li>1% Agarosegel & purification of the 2kb band -> new backbone</li>
 +
<li>repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li>
 +
<li>Transformation 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:RFPcd/ pSBIK3:BFPcd </li>
 +
<li>Testing of the fluorescent proteins RFRP and BFP with the Teacan</li>
 +
<li>Colony PCR of pSBIC3:dnaQ with VR/VF</li>
 +
<li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li>
 +
</ul>
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</div>
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<br>
  
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iGEM Logo
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<li>Primer design for genesynthesis</li>
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</div>
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<li>Plasmid isolation of the overnight cultures</li>
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<div class="col-md-6 spacer_bottom_10px">
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<li>Restriction of pSBIC3:dnaQ, mCherry, E2050-cds</li>
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Irgentwas
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<li>Sequencing of dnaQ clones with VR/VF</li>
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</div>
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<li>Colony-PCR with CH34/VR on ugi-cda1</li>
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</div>
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<li>Sequencing of ugi-cda1</li>
 +
<li>Assembly of dnaQ, dam, seqA</li>
 +
<ul>
 +
<li>Q5 PCR</li>
 +
<ul>
 +
<li>template pSBIC3:dnaQ, primer CH35/CH36, tmp = 59.9&deg;</li>
 +
<li> template pSBIC3:dam-seqA, primer CH37/CH38, tmp = 58.3&deg;C</li>
 +
<li> template pSBIC3:dam-seqA, primer CH39/CH40, tmp = 57&deg;</li>
 +
<li>template pHSG-WTPolI, primer CH42/CH43, tmp = 58.4&deg;C</li>
 +
<li>template pHSG-EPPolI, primer CH42/CH43, tmp = 58.4&deg;C </li>
 +
</ul>
 +
<li>DpnI digestion, gel extraction</li>
 +
<li>Restriction of pSBIC3:RFPcd with Xba/SpeI, gel extraction of 2 kb band</li>
 +
</ul>
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</ul>
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</div>
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<br>
  
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<ul>
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<li>Gibson of dnaQ, dam, seqA, C3-BB</li>
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<li data-target="#myCarousel" data-slide-to="2"></li>
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<li>Transformation in KRX compis</li>
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</ol>
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<li>Colony-PCR with the Gibson clones -> no bands</li>
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<li>Q5 PCR</li>
 +
<ul>
 +
<li>template pSBIC3:dnaQ, primer CH35/36, tmp = 59.9&deg;C</li>
 +
<li> template pSBIC3:dam-seqA, primer CH37/38, tmp = 58.3&deg;C </li>
 +
<li> template pSBIC3:dam-seqA, primer CH39/40, tmp = 57&deg;C </li>
 +
<li> template pSBIK3:J04450, primer CH30/31, tmp = 67&deg;C </li>
 +
</ul>
 +
<li>DpnI distriction and gel extraction of pSBIK3 backbone</li>
 +
<li>Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)</li>
 +
<li>Transformation in DH5&alpha;</li>
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</ul>
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</div>
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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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<li>colony-PCR of mutagenisis assembly I with VR/VF -> plating positive clones out</li>
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<div class="item">
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<li>Sequencing of positive clones</li>
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<img src="http://www.lonecke-zetel.de/uploads/pics/Farben_19.jpg" alt="B">
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<li>Q5 PCR</li>
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</div>
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<ul>
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<div class="item">
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<li>template pSBIC3:emrR, primer BF1/2</li>
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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> template pSBIC3:ugi-cda1, primer BF4/5</li>
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</div>
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<li> template pSBIC3:emrR, primer BF6/7</li>
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</div>
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<li> template pHSG:EPPolI, primer CH42/43</li>
 +
<li> template pHSG:WTPolI, primer CH42/43</li>
 +
</ul>
 +
<li>PCR clean up of the Q5 PCR</li>
 +
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3-BB (ASII)</li>
 +
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3-BB</li>
 +
<li>Transformation in DH5&alpha;</li>
 +
</ul>
 +
</div>
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<br>
  
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<button data-toggle="collapse" data-target="#18">Week 18: (01.08.16-07.08.16): </button>  
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<li>Colony PCR -> mutagen assembly II did not work</li>
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<li>Repeated colony PCR -> sequencing of positive clones</li>
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<nav class="navbar navbar-default custom_navbar_style">
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<button data-toggle="collapse" data-target="#19">Week 19: (08.08.14-14.08.16): </button>  
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<div id="navbar" class="navbar-collapse collapse">
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<li>Transformation of pSBIC3:K584001 & K608010</li>
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<li class="navbar_button active"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/2016_BielefeldCeBiTec_HomeTest">HOME</a></li>
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<li>Planning of reversion experiments</li>
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<a href="#">Official Team Profile</a>
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                                                <a href="#"> Members</a>
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                                                <a href="#"> Subteams</a>
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                                                <a href="#"> University Bielefeld</a>
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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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                                                </ul>
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<a href="#">Acknowledgement</a>
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<a href="#">Sponsors</a>
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<a href="#">Description</a>
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                                                <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Notebook"> Notebook</a>
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                                                <ul class="dropdown-extra">
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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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<li class="navbar_button"><a href="#">AWARDS</a></li>
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</ul>
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</div>
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</div>
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</nav>
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        <div class="maincontentOfpage">
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            <h1>Lab Notebook Mutation</h1>
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<div class="thumbnail">
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<h2>Lab-Notebook 06.08.2016</h2>
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<h3>PCR product for assembly of RFP expression template</h3>
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−
Amplification of RBS-RFP-Terminator insert from pSB1C3:E1010
+
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<table class="table table-hover">
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−
<colgroup> <col width=35%> <col width=65%> </colgroup>
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−
<tbody>
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−
<tr>
+
−
<td>polymerase</td>
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−
<td>Q5</td>
+
−
</tr>
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−
<tr>
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−
<td><i>for</i>-Primer</td>
+
−
<td>CH22 RBS_RFP_Ter_for</td>
+
−
</tr>
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−
<tr>
+
−
<td><i>rev</i>-Primer</td>
+
−
<td>CH23 RBS_RFP_Ter_rev</td>
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−
</tr>
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−
<tr>
+
−
<td>template</td>
+
−
<td>pSB1C3::E1010</td>
+
−
</tr>
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−
<tr>
+
−
<td>Tm</td>
+
−
<td>59 &deg;C</td>
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−
</tr>
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−
<tr>
+
−
<td>elongation time</td>
+
−
<td>24 sec</td>
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−
</tr>
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−
</tbody>
+
−
</table>
+
−
Amplification of the pSB1C3-backbone with addition of different Anderson promoters
+
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<table class="table table-hover">
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<colgroup> <col width=35%> <col width=65%> </colgroup>
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−
<tbody>
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−
<tr>
+
−
<td>polymerase</td>
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−
<td>Q5</td>
+
−
</tr>
+
−
<tr>
+
−
<td><i>for</i>-Primer</td>
+
−
<td>CH24 C3BB_Ter_for</td>
+
−
</tr>
+
−
<tr>
+
−
<td><i>rev</i>-Primer</td>
+
−
<td>CH25/26/27 C3BB_And_X_rev (X=01/047/086)</td>
+
−
</tr>
+
−
<tr>
+
−
<td>template</td>
+
−
<td>pSB1C3::E1010</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Tm</td>
+
−
<td>64 &deg;C</td>
+
−
</tr>
+
−
<tr>
+
−
<td>elongation time</td>
+
−
<td>1:07 min:sec</td>
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−
</tr>
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−
</tbody>
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−
</table>
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−
</div>
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−
        </div>
+
  
 +
<button data-toggle="collapse" data-target="#20">Week 20: (15.08.16-21.08.16): </button>
 +
<div id="20" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Sequencing results: mutagenesis AS I has mutations, AS II did not work</li>
 +
<li>Transformation of ASII Gibson</li>
 +
<li>Plasmid isolation of ASI/ ASII clones</li>
 +
<li>Colony PCR of ASII</li>
 +
<li>Restriction of positive clones of ASII</li>
 +
<li>Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
 +
<li>Restriction of right ASII clones with Eco/PstI</li>
 +
<li>Restriction of right ASI clone with Eco/PstI</li>
 +
</ul>
 
</div>
 
</div>
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<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.12.4/jquery.min.js"></script>
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<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" 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>PCR for coloning ASI into pSBIC3</li>
 +
<ul>
 +
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, tmp = 58.6&deg;C</li>
 +
<li>template pSBIC3:RFPcd, primer CH30/31, tmp = 67&deg;C</li>
 +
<li>template pSBIC3:AraC-Pbad, primer CH54/52, tmp = 58.6&deg;C</li>
 +
</ul>
 +
<li>DpnI digestion</li>
 +
<li>Restriction with Eco/Pst</li>
 +
<ul>
 +
<li>pSBIK3:J04450</li>
 +
<li> pSBIC3:K608010</li>
 +
<li> pSBIC3:K584001</li>
 +
</ul>
 +
<li>Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP</li>
 +
<li>Restriction with EcoRI/PstI of  ASII and pSBIC3:Strong RFP</li>
 +
<li>Ligation of ASII and strong RFP</li>
 +
<li>Transformation of the ligation into DH5&alpha;</li>
 +
<li>Gel extraction of pSBIK3, K608010, K584001</li>
 +
<li>Ligation of pSBIK3 + K608010</li>
 +
<li>Overnight culture of the ligation</li>
 +
<li>Gel extraction of PCR products & PCR clean up</li>
 +
<li>Gibson assembly</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>Ligation & Transformation in DH5&alpha;</li>
 +
<li>Colony PCR</li>
 +
<ul>
 +
<li>template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, tmp = 58&deg;C</li>
 +
<li> template pSBIC3:ASI, primer VR/VF_rev, tmp = 58&deg;C </li>
 +
 
 +
</ul>
 +
<li>Q5 PCR</li>
 +
<ul>
 +
<li> template A68T_A, primer CH20/A68T_rev, tmp = 58.6&deg;C (AA)</li>
 +
<li> template A68T_B, primer A68T_B/CH21, tmp = 58.5&deg;C (AB)</li>
 +
<li> template C379A_A, primer CH20/C379A_rev, tmp = 57.7&deg;C (BA)</li>
 +
<li> template C379A_B, primer C379A_fw/CH21, tmp = 58&deg;C (BB)</li>
 +
<li> template A380T_A, primer CH20/ A380T_rev, tmp = 58.1&deg;C (CA)</li>
 +
<li> template A380T_B, primer A380T_fw/CH21, tmp = 58.1&deg;C (CB)</li>
 +
</ul>
 +
<li>Gel extraction</li>
 +
<li>Gibson assembly</li>
 +
<li>Transformation in KRX</li>
 +
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
 +
<li>Colony PCR of AraC-Pbad (non leaky), ASI</li>
 +
<li>Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, tmp = 57.5&deg;C</li>
 +
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
 +
<li>Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, tmp = 50&deg;C</li>
 +
<li>Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
 +
<li>Restriction of C3 backbone with Eco/Pst</li>
 +
<li>Transformation of pSBIC3:K516031 in KRX</li>
 +
<li>cPCR of pSBIC3:K608010_StopA/B/C with VR/VF</li>
 +
<li>Sequencing A with VF, B with VR and C with VR</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#22">Week 22: (29.08.16-04.09.16): </button>
 +
<div id="22" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Plasmid isolation of Stop-GFPs -> Sequencing</li>
 +
<li>Restriction</li>
 +
<ul>
 +
<li>pSBIC3:AraCPbad with SpeI, PstI</li>
 +
<li> pSBIC3:AraCPbad(non-leaky) with SpeI, PstI </li>
 +
<li> pSBIC3:RFP Generator with Xba, PstI </li>
 +
<li> pSBIK3:MP6-I-Klon 4 with SpeI, PstI </li>
 +
<li> pSBIK3:MP6-II-Klon with Xba, PstI </li>
 +
</ul>
 +
<li>Designing of new primer</li>
 +
<li>Gel extraction of the Restriction</li>
 +
<li>Ligation </li>
 +
<ul>
 +
<li>C3:AraC-Pbad + RFP Gen</li>
 +
<li>C3:AraC-Pbad(non-leaky) + RFP Gen</li>
 +
<li>K3:Mut-I + Mut II</li>
 +
<li>C3 + MutII</li>
 +
</ul>
 +
<li>Repeat of Restriction of ASII: template K3:ASII, EcoRI-Hf/PstI</li>
 +
<li>Gel extraction of the restriction</li>
 +
<li>Ligation</li>
 +
<li>Transformation of the Ligation, pHSG-EPPolI, pHSG-WTPolI in KRX</li>
 +
<li>cPCR with VR/VR 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>Q5 PCR, tmp = 57&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>DpnI digestion</li>
 +
<li>Gel extraction</li>
 +
<li>Gibson assembly</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>Transformation</li>
 +
<li>cPCR with VR/VF, tmp = 57&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>Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#23">Week 23: (05.09.16-11.09.16): </button>
 +
<div id="23" class="collapse">  
 +
<br>
 +
<ul>
 +
<li>cPCR of ugi-cda1 -> Sequencing of right clones</li>
 +
<li>Q5 PCR, tmp = 58&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>DpnI digestion of the Q5 PCR</li>
 +
<li>Gel extraction of Stop A, Stop B</li>
 +
<li>Gibson assembly stop A + stop B</li>
 +
<li>Restriction with Eco/Pst ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
 +
<li>Transformation of C3:PRha, C3:PRha-GFPGen in KRX</li>
 +
<li>Transformation of PRha-GFPGen in top10</li>
 +
<li>Transformation of StoppA+B in DH5&alpha;</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#24">Week 24: (12.09.16-18.09.16): </button>
 +
<div id="24" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Q5 PCR, tmp = 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>DpnI digestion</li>
 +
<li>Gel extraction</li>
 +
<li>Gibson assembly of seqA</li>
 +
<li>Transformation in DH5&alpha; of C3:SeqA, K3:StopA+B</li>
 +
<li>Q5 PCR of C3:damSeqA with CH71/72, tmp = 58&deg;C</li>
 +
<li>DpnI digestion</li>
 +
<li>Gibson assembly< of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
 +
<li>Transformation of the Gibson assembly 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>cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR</li>
 +
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
 +
<li><Q5 PCR of C3:damSeqA, primer 71/72, gradient 55-59&deg;C/li>
 +
<li>Sequencing of stop A+B</li>
 +
<li>Restriction of K3:ASII, C3:Terminator with EcoRI, PstI</li>
 +
<li>Gibson assembly of M6 in RFPGen -> Transformation</li>
 +
<li>Test cultivation of a 96-well plate</li>
 +
<li>cPCR of M6-Gen -> Plasmid isolation</li>
 +
<li>Restriction of dnaQ-Gen with Xba/Pst</li>
 +
<li>araE-PCR with tmp 58&deg;C</li>
 +
<li>Gibson assembly of araE</li>
 +
<li>PCR of DNA, primer 81/82 and C3:med RFP 83/84</li>
 +
<li>Gel extraction of dnaQ-Gen</li>
 +
<li>Ligation with C3:AraC-Pbad/C3:PRha</li>
 +
<li>DpnI digestion</li>
 +
<li>PCR clean up</li>
 +
<li>Gibson of araE-Insert + araE-BB</li>
 +
<li>Transformation of the ligation in KRX</li>
 +
<li>Restriction of C3:M6-Gen with Eco/Xba, weak/RFP Gen with Xba/Pst</li>
 +
<li>cPCR of C3:araE-Device</li>
 +
<li>Gibson C3 BB + ugi_cda1, C3 BB + ASII</li>
 +
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
 +
<li>cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF</li>
 +
</ul>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#25">Week 25: (19.09.16-25.09.16): </button>
 +
<div id="25" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Restriction of M6-Generator with Xba/Pst, C3:K808000, C3:Prha with Spe/Pst</li>
 +
<li>Clean up</li>
 +
<li>Ligation with BB</li>
 +
<li>Transformation of Ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
 +
<li> Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&deg;C</li>
 +
<li>Transformation 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; 1-2h growth, +25mM arabinose/25mM glycose</li>
 +
<li>Transformation of JS200 EP/WT with plA230</li>
 +
<li>cPCR of AraC-Pbad-M6; Prha-M6</li>
 +
<li>Transformation of plA230 in JS00 EP/WT</li>
 +
<li>Rifampicillin assay</li>
 +
<li>beta-lactamase Reversions assay</li>
 +
<li>Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10</li>
 +
<li>Overnight cultures</li>
 +
</ul>
 +
<br>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#26">Week 26: (26.09.16-02.10.16):</button>
 +
<div id="26" class="collapse">
 +
<br>
 +
<ul>
 +
<li>Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + plA230 in Top10</li>
 +
<li>Transformation 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>Restriction 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>Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 +
<li>Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 +
<li>Reversions assay</li>
 +
<li>Restriction of E/P of pSB4C5(E/P)</li>
 +
<li>Q5 PCR</li>
 +
<ul>
 +
<li>template C3:ugi-cda, primer 67/68, tmp = 58&deg;C</li>
 +
<li>template C3:cda, primer 69/70, tmp = 60&deg;C</li>
 +
<li>template A3, primer CK06/05, tmp = 59.9&deg;C</li>
 +
<li>template reporter, primer CK07/08, tmp = 59&deg;C</li>
 +
 
 +
</ul>
 +
</ul>
 +
<br>
 +
</div>
 +
<br>
 +
 
 +
<button data-toggle="collapse" data-target="#27">Week 27: (03.10.16-09.10.16): </button>  
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<li>Transformation of C3:dnaQ Dev + plA230, C3:M6 Dev + plA230 in Top10</li>
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<li>Analysis reversion assay</a>
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Revision as of 02:23, 19 October 2016



lab notebook.

Mutation


Firstly, we researched for literature on issues involving in vivo mutagenesis and error prone polymerase. In this phase we read the paper from [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.
Platting provided JS200 strain out.


  • Plasmid isolation of:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • psBIK3:J04450
    • Overnight cultures from JS200.
    • JS200 glycerin stocks (500 μ culture + 200 μ 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 & restriction with SpeI.
  • Gel
  • Idea: BioBrick site directli behind pMBi ori of psBIK3
    • 1. Deletion of BioBrick site
      • Q5 PCR
        • Template: pSBIK3:J04450
        • part 1 with primer CH07/CH21, tm=56°C
        • part 2 with primer CH08/CH20, tm=57°C
        • -> part 1 worked; part 2 not
      • Transformation in DH5α of pSBIC3:K592100
    • 2.: New BioBrick site behind ori


  • Restriction of pSBIK3:J04450 with SPEI and Xba
  • colony PCR of K592100
  • gradient PCR of part 1 & part 2


  • 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 μ Gibson Mastermix + 3.5 μ pSBIK3 Backbone + 1.5 μ 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 with EcoRI/ PstI
  • 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, tm = 67 °C
  • DpnI digestion of pcr product pSBIC3:K516132, 1h 37°C
  • 1% Agarosegel & purification of the 2kb 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:RFPcd/ pSBIK3:BFPcd
  • Testing of the fluorescent proteins RFRP 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 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 CH35/CH36, tmp = 59.9°
      • template pSBIC3:dam-seqA, primer CH37/CH38, tmp = 58.3°C
      • template pSBIC3:dam-seqA, primer CH39/CH40, tmp = 57°
      • template pHSG-WTPolI, primer CH42/CH43, tmp = 58.4°C
      • template pHSG-EPPolI, primer CH42/CH43, tmp = 58.4°C
    • DpnI digestion, gel extraction
    • Restriction of pSBIC3:RFPcd with Xba/SpeI, gel extraction of 2 kb band


  • Gibson of dnaQ, dam, seqA, C3-BB
  • Transformation in KRX compis
  • Colony-PCR with the Gibson clones -> no bands
  • Q5 PCR
    • template pSBIC3:dnaQ, primer CH35/36, tmp = 59.9°C
    • template pSBIC3:dam-seqA, primer CH37/38, tmp = 58.3°C
    • template pSBIC3:dam-seqA, primer CH39/40, tmp = 57°C
    • template pSBIK3:J04450, primer CH30/31, tmp = 67°C
  • DpnI distriction and gel extraction of pSBIK3 backbone
  • Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)
  • Transformation in DH5α


  • colony-PCR of mutagenisis assembly I 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-BB (ASII)
  • Gibson assembly of EPPolI/ WTPolI in pSBIK3-BB
  • Transformation in DH5α


  • Colony PCR -> mutagen assembly II did not work
  • Repeated colony PCR -> sequencing of positive clones


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


  • Sequencing results: mutagenesis AS I has mutations, AS II did not work
  • Transformation of ASII Gibson
  • Plasmid isolation of ASI/ ASII clones
  • Colony PCR of ASII
  • Restriction of positive clones of ASII
  • Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR
  • Restriction of right ASII clones with Eco/PstI
  • Restriction of right ASI clone with Eco/PstI


  • Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)
  • PCR for coloning ASI into pSBIC3
    • template pSBIK3:dnaq/dam/seqA, primer CH35/40, tmp = 58.6°C
    • template pSBIC3:RFPcd, primer CH30/31, tmp = 67°C
    • template pSBIC3:AraC-Pbad, primer CH54/52, tmp = 58.6°C
  • DpnI digestion
  • Restriction with Eco/Pst
    • pSBIK3:J04450
    • pSBIC3:K608010
    • pSBIC3:K584001
  • Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
  • Restriction with EcoRI/PstI 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, tmp = 58°C
    • template pSBIC3:ASI, primer VR/VF_rev, tmp = 58°C
  • Q5 PCR
    • template A68T_A, primer CH20/A68T_rev, tmp = 58.6°C (AA)
    • template A68T_B, primer A68T_B/CH21, tmp = 58.5°C (AB)
    • template C379A_A, primer CH20/C379A_rev, tmp = 57.7°C (BA)
    • template C379A_B, primer C379A_fw/CH21, tmp = 58°C (BB)
    • template A380T_A, primer CH20/ A380T_rev, tmp = 58.1°C (CA)
    • template A380T_B, primer A380T_fw/CH21, tmp = 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, tmp = 57.5°C
  • Sequencing of pSBIC3:emrR-1/-2 with VR/VF
  • Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, tmp = 50°C
  • Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010
  • Restriction of C3 backbone with Eco/Pst
  • Transformation of pSBIC3:K516031 in KRX
  • cPCR 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
    • pSBIC3:AraCPbad with SpeI, PstI
    • pSBIC3:AraCPbad(non-leaky) with SpeI, PstI
    • pSBIC3:RFP Generator with Xba, PstI
    • pSBIK3:MP6-I-Klon 4 with SpeI, PstI
    • pSBIK3:MP6-II-Klon with Xba, PstI
  • Designing of new primer
  • Gel extraction of the Restriction
  • Ligation
    • C3:AraC-Pbad + RFP Gen
    • C3:AraC-Pbad(non-leaky) + RFP Gen
    • K3:Mut-I + Mut II
    • C3 + MutII
  • Repeat of Restriction of ASII: template K3:ASII, EcoRI-Hf/PstI
  • Gel extraction of the restriction
  • Ligation
  • Transformation of the Ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
  • cPCR with VR/VR 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, tmp = 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
  • DpnI 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
  • cPCR with VR/VF, tmp = 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


  • cPCR of ugi-cda1 -> Sequencing of right clones
  • Q5 PCR, tmp = 58°C
    • template C3:damSeqA, primer CH71/72
    • template C3:StoppGFP Stop A Stop B, primer CH20/66
    • template C3:damSeqA, primer CH73/74
    • template K3:Stopp GFP A+B, primer CH64/21
  • DpnI digestion of the Q5 PCR
  • Gel extraction of Stop A, Stop B
  • Gibson assembly stop A + stop B
  • Restriction with Eco/Pst ASI+ASII, GFP_Astop_2, GFP_Astop_2
  • Transformation of C3:PRha, C3:PRha-GFPGen in KRX
  • Transformation of PRha-GFPGen in top10
  • Transformation of StoppA+B in DH5α


  • Q5 PCR, tmp = 58°C
    • template C3:dnaQ, primer 54/55
    • template C3:WTPolI (CWP1), primer 58/59
    • template C3:EPPolI (CEP1), primer 58/59
    • template C3:ASI+ASII, primer 54/76
    • template C3:K516031, primer 56/57
    • template C3: K516031, primer 60/61
    • template C3: K516031, primer 60/61
    • template C3: K516031, primer 75/57
  • DpnI digestion
  • Gel extraction
  • Gibson assembly of seqA
  • Transformation in DH5α of C3:SeqA, K3:StopA+B
  • Q5 PCR of C3:damSeqA with CH71/72, tmp = 58°C
  • DpnI digestion
  • Gibson assembly< of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB
  • Transformation of the Gibson assembly in DH5α
  • Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
  • Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
  • cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR
  • Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter
  • Sequencing of stop A+B
  • Restriction of K3:ASII, C3:Terminator with EcoRI, PstI
  • Gibson assembly of M6 in RFPGen -> Transformation
  • Test cultivation of a 96-well plate
  • cPCR of M6-Gen -> Plasmid isolation
  • Restriction of dnaQ-Gen with Xba/Pst
  • araE-PCR with tmp 58°C
  • Gibson assembly of araE
  • PCR of DNA, primer 81/82 and C3:med RFP 83/84
  • Gel extraction of dnaQ-Gen
  • Ligation with C3:AraC-Pbad/C3:PRha
  • DpnI digestion
  • PCR clean up
  • Gibson of araE-Insert + araE-BB
  • Transformation of the ligation in KRX
  • Restriction of C3:M6-Gen with Eco/Xba, weak/RFP Gen with Xba/Pst
  • cPCR of C3:araE-Device
  • Gibson C3 BB + ugi_cda1, C3 BB + ASII
  • Transformation of the Gibson assembly in DH5α
  • cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF


  • Restriction of M6-Generator with Xba/Pst, C3:K808000, C3:Prha with Spe/Pst
  • Clean up
  • Ligation with BB
  • 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; 1-2h growth, +25mM arabinose/25mM glycose
  • Transformation of JS200 EP/WT with plA230
  • cPCR 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 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 of E/P of pSB4C5(E/P)
  • Q5 PCR
    • template C3:ugi-cda, primer 67/68, tmp = 58°C
    • template C3:cda, primer 69/70, tmp = 60°C
    • template A3, primer CK06/05, tmp = 59.9°C
    • template reporter, primer CK07/08, tmp = 59°C



  • Transformation of C3:dnaQ Dev + plA230, C3:M6 Dev + plA230 in Top10
  • Reversions assay


  • Analysis reversion assay