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| | {{Team:Berlin/AllCss}} | | {{Team:Berlin/AllCss}} |
| - | {{Team:Berlin/scrollNav}}
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| | <html lang="en"> | | <html lang="en"> |
| - | <script type="text/javascript">
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| | <div id="subside-content"> | | <div id="subside-content"> |
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| - | | + | <div class="row"> |
| | + | <div class="col-3-xs"> |
| | + | <div class="container"> |
| | + | <h1>One</h1> |
| | + | <h1>Two</h1> |
| | + | <h1>Three</h1> |
| | + | </div> |
| | + | </div> |
| | + | <div class="col-9-xs"> |
| | <!--- ////////////////// DESCRIPTION////////////////// --> | | <!--- ////////////////// DESCRIPTION////////////////// --> |
| | <div class="container"> | | <div class="container"> |
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| | </div> | | </div> |
| | </div> | | </div> |
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| - | <div class="row">
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| - | <div class="col-md-3 col-sm-3 col-xs-3">
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| - | <div class="well bs-sidebar affix" id="sidebar">
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| - | <ul class="nav nav-pills nav-stacked">
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| - | <li><a href="#">Link 1</a></li>
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| - | <li><a href="#">Link 2</a></li>
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| - | </ul>
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| - | </div> <!--well bs-sidebar affix-->
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| - | </div> <!--col-md-3-->
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| | <div class="row"> | | <div class="row"> |
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| | </div> | | </div> |
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| - | <!--- ////////////////// Results ////////////////// -->
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| - | <div class="container">
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| - | <div class="row hidden-xs">
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| - | <div class="col-xs-12">
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| - | <div class="project-entry-tags">
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| - | <ul>
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| - | <li><a href="#top">GO TO TOP</a></li>
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| - | </ul>
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| - | <div class="row">
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| - | <div class="col-xs-12">
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| - | <div class="project-number">5</div><div class="project-headline-float"><h2 class="green-text project-headline"> The Results</h2></div>
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| - | </div>
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| - | </div>
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| - |
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| - | <div class="row">
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| - | <!--<div class="col-sm-4 hidden-xs">
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| - | <img src="img/blog/Team_Berlin_bacteria.png" class="img-responsive" style="margin: 0 auto;"/>
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| - | </div>-->
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| - | <div class="col-xs-12 col-sm-12 blog-text" style="margin-bottom:40px;">
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| - | <p>
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| - | <a name="results"> </a>
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| - | Results, jo!
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| - | </div>
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| - | </div>
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| - | </div>
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| | | | |
| - | <!--- ////////////////// Lab Diary ////////////////// -->
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| - | <div class="container">
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| - | <div class="row hidden-xs">
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| - | <div class="col-xs-12">
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| - | <div class="project-entry-tags">
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| - | <li><a href="#top">GO TO TOP</a></li>
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| - | <div class="row">
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| - | <div class="col-xs-12">
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| - | <div class="project-number">6</div><div class="project-headline-float"><h2 class="green-text project-headline"> Lab Summary</h2></div>
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| - | </div>
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| - | </div>
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| - |
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| - | <div class="row">
| |
| - | <!--<div class="col-sm-4 hidden-xs">
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| - | <img src="img/blog/Team_Berlin_bacteria.png" class="img-responsive" style="margin: 0 auto;"/>
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| - | </div>-->
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| - | <div class="col-xs-12 col-sm-12 blog-text" style="margin-bottom:40px;">
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| - | <p>
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| - | <a name="lab-summary"> </a>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 1: 02.04.2014 – 06.04.2014</h2>
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| - | Cultivation of E. coli Nissle 1917<br/>
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| - |
| |
| - | Genomic DNA extraction of E.coli Nissle strain<br/>
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| - |
| |
| - | Production of BfR, FTNA1 and FTNA2<br/>
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| - | Restriction digest<br/>
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| - | PCR Purification<br/>
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| - | Ligation Assay<br/>
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| - | Transformation into DH5α-Cells<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 2: 07.04.2014 – 13.04.2014</h2>
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| - | Colony PCR to check the results<br/>
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| - |
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| - | Bfr, FTNA 1, FTNA 2 primar designed for amplification<br/>
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| - |
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| - | Transformation of pQE_80L into DH5α<br/>
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| - |
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| - | Cultivation of BfR, FTNA 1, FTNA 2 in LB<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 3: 14.04.2014 – 20.04.2014</h2>
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| - | Miniprep of the cells from Week 2 <br/>
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| - |
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| - | DNA concentration determination and sequencing<br/>
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| - |
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| - | Expression of BfR, FTNA 1, FTNA 2 and induction with IPTG<br/>
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| - |
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| - | Production of a Mutaflor-Supression culture and streaking<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 4: 21.04.2014 – 27. 04.2014 </h2>
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| - | Minirep
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 5: 28.04.2014 – 04.05.2014</h2>
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| - | PCR Plasmid/Primar and Parameter check with Q5 Polymerase and Phusion Polymerase<br/>
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| - |
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| - | Miniprep of [pKD46+ DH5α] and [pKD4 + DH5 α]<br/>
| |
| - | Genomic DNA extraction from Pseudomonas putida<br/>
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| - | Enzyme digestion of different plasmids<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 6: 05.05.2014 – 11.05.2014</h2>
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| - | Preculture of strains from Budisa strain databse in LB medium and Midiprep<br/>
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| - |
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| - | Canamycin cassette PCR of pKD4<br/>
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| - | Restriction digest of Plasmid pKD4 and pKD46v<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 7: 12.05.2014 – 18.05.2014</h2>
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| - | Biotransformation of Ferritin and pKD46 in Nissle and DH5 α strains
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 8: 19.05.2014 – 25.05.2014</h2>
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| - | Gel extraction of FieF PCR<br/>
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| - |
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| - | Gene Knockout of FieF in [RV + pKD46] and [WM10+pKD46]<br/>
| |
| - |
| |
| - | Transformation of pKD46 in Nissle and MG 1655<br/>
| |
| - |
| |
| - | PCR of ATPCS and PPMT<br/>
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| - |
| |
| - | Digestion of pQE_80L for cloning<br/>
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| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 9: 26.05.2014 – 01.06.2014</h2>
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| - | Production of LB plates<br/>
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| - |
| |
| - | AMB-1 and Microfluidic Chip were picked up from Max-Plank Institute<br/>
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| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 10: 02.06.2014 – 08.06.2014</h2>
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| - | Colony PCR and analytical gel electrophoresis for identifying the right clones<br/>
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| - |
| |
| - | PCR of ATPCS and PPMT to identify the correct amplification parameter.<br/>
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| - |
| |
| - | Colony PCR for Knockout.<br/>
| |
| - |
| |
| - | PCR of ATPCS and PPMT with Q5 High Fidelity Mastermix.<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 11: 09.06.2014 – 15.06.2014</h2>
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| - | Refine the PCR of PPMT and ATPCS with DMSO<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 12: 16.06.2014 – 22.06.2014</h2>
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| - | PCR amplification of ATPCS from cDNA<br/>
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| - | Colony PCR to seperate cells with FieF Knockout<br/>
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| - | Restrcition digest of pQE_80L and ATPCS-PCR Fragment<br/>
| |
| - | Test expression of Ferritin in RV308 (pSB1C3_Ferritin) and Nissle (pSB1C3_Ferritin)<br/>
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| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 13: 23.06.2014 – 29.06.2014</h2>
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| - | Amplification of ATPCS from cDNA and PCR Purification<br/>
| |
| - | Preparation of Nissle, Nissle + Ferritin, RV 308, Rv 308 + Ferritin pre-cultures<br/>
| |
| - | Transformation of human Ferritin on PC514 to Nissle and RV 308<br/>
| |
| - | Induction of Ferritin expression with IPTG<br/>
| |
| - | </div>
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| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline">Week 14: 30.06.2014 – 06.07.2014</h2>
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| - | Colony PCR and Ligation of ATPCS<br/>
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| - | Knockout of FieF and FUR in RV308 and Nissle<br/>
| |
| - | Transformation of RFP device and Ferritin in RV308, Nissle and WM110 strains.<br/>
| |
| - | PCR amplification of knockout cassette FUR/FieF<br/>
| |
| - | Expression of RV308 (RFP) and RV308 (RFP + Ferritin) after Induction with IPTG<br/>
| |
| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 15: 07.07.2014 – 13.07.2014</h2>
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| - | Preparation of cryostocks and pre-cultures of ATPCS clone 1-10, WM 110, RV 308, Nissle<br/>
| |
| - | PCR of PPMT with goTaq Polymerase<br/>
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| - | Miniprep of ATPCS clone number 3<br/>
| |
| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 16: 14.07.2014-20.07.2014</h2>
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| - | Midiprep of pQE_80L , PMA-T_PPMT, pKD46 <br/>
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| - | Digestion of pQE_80L with Hind III and SacI<br/>
| |
| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 17: 21.07.2014-27.07.2014</h2>
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| - | Miniprep and Restriction of pBADex-mYFP Venus (Amp); pEx-HisII (Amp); pJS418_Phagemid (dummy) (Cm)<br/>
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| - | Ligation of PPMT Fragment from pMAC_PPMT into pEX_HisII<br/>
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| - | </div>
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 18: 28.07.2014-3.08.2014</h2>
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| - | Degradation test of prepped TB-Expression Plasmids <br/>
| |
| - | Transformation of pEX_His_PPMT in MG1655, DH5α, DH10B strains<br/>
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| - | Colony PCR of the PPMT clones<br/>
| |
| - | Miniprep and Sequencing of pEX_His_PPMT in DH5 α<br/>
| |
| - | Ligation of ATPCS PCR Fragment into pQE_80 L<br/>
| |
| - | Colony PCR of pQE_80L_ATPCS<br/>
| |
| - | </div>
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline">Week 19: 04.08.2014 -10.08.2014</h2>
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| - | Preculture of ATPCS clones for Sequencing <br/>
| |
| - | PCR of BamHI_PPMT_GS, GS_ATPCS_HindIII, BamHi_HuFerritin_HindIII<br/>
| |
| - | Transformation of BFR M52H in DH10B.<br/>
| |
| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 20: 11.08.2014 - 17.08.2014</h2>
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| - | Prepare chemically competent cells<br/>
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| - | Generation of Heme-free BFR by Site-directed Mutagenesis<br/>
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| - | Digestion of various PCR fragements for cloning into pQE_80L<br/>
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| - | Colony PCR of pQE_80L<br/>
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| - | Clone Sequencing<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 21: 18.08.2014 – 24.08.2014</h2>
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| - | SDS-PAGE with Coomasie staining for identification of protein expression<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 22: 25.08.2014 – 31.08.2014</h2>
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| - | Calibration curve for iron concentration measurement<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 23: 15.09.2014 – 21.09.2014</h2>
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| - | Preparing cultures for fluorescence microscopy<br/>
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| - | Constructing pQE_80L_T5_ATPCS_lac_PPMT<br/>
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| - | Digest and Dephosphorylation of vector pQE_80L_ATPCS<br/>
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| - | PCR of Biobrick parts (BB0-BB3)<br/>
| |
| - | Digest of pSB1C3-Ferritin<br/>
| |
| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 24: 22.09.2014 – 28.09.2014</h2>
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| - | Preparation of pre-cultures<br/>
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| - | Checking insertion of gene in ATPCS_PPMT clones by colony PCR<br/>
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| - | Digestion of miniprepped pSB1C3_Ferritin (Calgary) for extraction of vector for Biobrick preparation<br/>
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| - | Mutagenesis of ATPCS in different plasmid_ATPCS construct<br/>
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| - | Berlin Vector – pQE-80L-JBFS- huFerritin Assembly PCR<br/>
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| - | Isolation of plasmid DNA of pQE80L_ATPCSMut_GS_PPMT and pQE80L_ATPCS_PPMT <br/>
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| - | Repeat PCR of Biobricks<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 25: 29.09.2014 – 05.10.2014</h2>
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| - | Digestion of Biobrick parts<br/>
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| - | Ligation of Biobrick parts with pSB1C3 backbone<br/>
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| - | Plasmid Digestion and checked with PCR<br/>
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">Week 25 06.10.2014 – 12.10.2014</h2>
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| - | Colony PCR<br/>
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| - | Miniprep of cultures and preparation of sequencing samples<br/>
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| - | Preculture of knockouts<br/>
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| - | PCR of pSB1C3 backbone<br/>
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| - | Extraction of PCR Product (pSB1C3 linearised)<br/>
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| - | Iron concentration measurement in knockout strains<br/>
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| - | </div>
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| - | </div>
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| - | </div>
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| - | </div>
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| - |
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| - | <!--- ////////////////// Notebook////////////////// -->
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| - | <div class="container">
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| - | <div class="row hidden-xs">
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| - | <div class="col-xs-12">
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| - | <div class="project-entry-tags">
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| - | <ul>
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| - | <li><a href="#top">GO TO TOP</a></li>
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| - | </ul>
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| - | </div>
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| - | </div>
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| - | </div>
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| - |
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| - | <div class="row">
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| - | <div class="col-xs-12">
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| - | <div class="project-number">7</div><div class="project-headline-float"><h2 class="green-text project-headline"> Notebook</h2></div>
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| - | </div>
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| - | </div>
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| - |
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| - | <div class="row">
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| - | <!--<div class="col-sm-4 hidden-xs">
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| - | <img src="img/blog/Team_Berlin_bacteria.png" class="img-responsive" style="margin: 0 auto;"/>
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| - | </div>-->
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| - | <div class="col-xs-12 col-sm-12 blog-text" style="margin-bottom:40px;">
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| - | <p>
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| - | <a name="notebook"> </a>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline">04/02 Wednesday - Cultivating E. coli Nissle 1917</h2>
| |
| - | LB plates were produced without antibiotic. Therefore 10 ml MQ-H<sub>2</sub>O were sterile filtered in 15 ml falcon tube. 1 capsule Mutaflor 331800 was solved in the sterile water.
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| - | The E. coli Nissle solution was incubated at 37°C and 200 rpm for 1 h and crossed out at 4 plates to get different dilution of cells.
| |
| - | <!--{| class="wikitable"
| |
| - | |-
| |
| - | ! dilution !! E. coli Nissle solution !! H<sub>2</sub>O !! dilution factor
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| - | |-
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| - | | I || 2 µl || 1998 µl || 1:10<sup>3</sup>
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| - | |-
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| - | | II || 100 µl from dilution I || 900 µl ||1:10<sup>4</sup>
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| - | |-
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| - | | III || 10 µl from dilution II || 990 µl || 1:10<sup>5</sup>
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| - | |}-->
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| - | </div>
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline"> 04/03 Thursday - Genomic DNA extraction of ECN - first step </h2>
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| - |
| |
| - | For extraction of the genomic DNA of E. coli Nissle, 2 E. coli colonies were oicked from LB plate (dilution 1:10<sup>3</sup>) and used for inocculation of 6 ml LB precultures. These were grown over night at 37°C and 200 rpm.
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| - | </div>
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| - |
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| - |
| |
| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline"> 04/04 Friday - Genomic DNA extraction of ECN - second step</h2>
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| - |
| |
| - | For preperation of the genomic DNA 1 ml of each preculture was taken and a genomic extraction performed using the Wizard Genomic DNA purification Kit. See instructions of the purification Kit by Promega. Genomic DNA was stored at 12°C in fridge.
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| - |
| |
| - | As E. coli Nissle is a natural organism without resistence genes both precultures were streaked out on LB, LB+KAN and LB+AMP. All plates were incuvated over the weekend at 37°C.
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| - |
| |
| - | DNA concentration was meassured by 260 nm using Goldi. DNA was diluted 1:40 in a UV cuvette and DNA absorption meassured at 260/28 nm in goldi.<br/>
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| - |
| |
| - | '''For genomic DNA:'''
| |
| - | {| class="wikitable"
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| - | |-
| |
| - | | 1: || 1390 ng/µl
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| - | |-
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| - | | 2: || 988 ng/µl
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| - | |}
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| - |
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| - | </div>
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| - |
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| - |
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| - | <div class="sub-content-project">
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| - | <h2 class="sub-content-project-headline"> 04/05 Saturday - Genomic DNA extraction of ECN - results </h2>
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| - |
| |
| - | Bacteria growth only on LB plate indicates that there was no contamination with AMP or KAN restinatant E. coli. This doesn´t mean that there is no contamination at all. See for sure on PCR.
| |
| - | <br/>
| |
| - | {| class="wikitable"
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| - | |-
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| - | ! LB !! LB+KAN !! LB+AMP
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| - | |-
| |
| - | | + || - || -
| |
| - | |}
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| - | </div>
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| - |
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| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/06 Sunday </h2>
| |
| - | ==Production of BfR, FTNA1 and FTNA2==
| |
| - | ===Restriction digest===
| |
| - | After 2 purifications of plasmids:
| |
| - | <br/>
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | p1 || 84ng/µl || 10µl
| |
| - | |-
| |
| - | | p2 || 72,2ng/µl || 10µl
| |
| - | |-
| |
| - | | p3 || 54,8ng/µl || 23µl
| |
| - | |}
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| - |
| |
| - | ====Restriction program====
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | || 1x || 4x
| |
| - | |-
| |
| - | | plasmid P2 || 3µl || 12µl
| |
| - | |-
| |
| - | | BamHI (FD) || 1µl || 4µl
| |
| - | |-
| |
| - | | HindIII (FD) || 1µl || 4µl
| |
| - | |-
| |
| - | | Buffer (FD) || 2µl || 8µl
| |
| - | |-
| |
| - | | H<sub>2</sub>O || 13µl || 52µl
| |
| - | |}
| |
| - |
| |
| - | --> 37°C for 1h (restriction)
| |
| - | --> 80°C for 10min (deactivation)
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | PCR || 6,38µl || 4,56µl || 2,71µl
| |
| - | |-
| |
| - | | BamHI (FD) || 1µl || 1µl || 1µl
| |
| - | |-
| |
| - | | HindIII (FD) || 1µl || 1µl || 1µl
| |
| - | |-
| |
| - | | Buffer (FD) || 2µl || 2µl || 2µl
| |
| - | |-
| |
| - | | H<sub>2</sub>O || 19,62µl || 21,44µl || 23,29µl
| |
| - | |}
| |
| - |
| |
| - | --> 37°C for 1h (restriction)
| |
| - | --> 80°C for 10min (deactivation)
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | Plasmid || 1 || 2 || 3
| |
| - | |-
| |
| - | | P || 10µl || 10µl || 18µl
| |
| - | |-
| |
| - | | BamHI (FD) || 1µl || 1µl || 1µl
| |
| - | |-
| |
| - | | HindIII (FD) || 1µl || 1µl || 1µl
| |
| - | |-
| |
| - | | Buffer (FD) || 2µl || 2µl || 3µl
| |
| - | |-
| |
| - | | nuc.free H<sub>2</sub>O || 6µl || 6µl || 7µl
| |
| - | |}
| |
| - |
| |
| - | --> 37°C for 1h (restriction)
| |
| - | --> 80°C for 10min (deactivation)
| |
| - |
| |
| - | ===PCR Purification (without isopropanol)===
| |
| - |
| |
| - | Elution buffer 20µl; 1min incubated
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | plasmid P1-3 || 45,3ng/µl
| |
| - | |-
| |
| - | | BfR || 12,9ng/µl
| |
| - | |-
| |
| - | | FTNA1 || 34,4 ng/µl
| |
| - | |-
| |
| - | | FTNA2 || 29,9ng/µl
| |
| - | |}
| |
| - |
| |
| - | ===Ligation Assay===
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! Control !! BFR !! BFR !! BFR !! FTNA1 !! FTNA1 !! FTNA1 !! FTNA2 !! FTNA2 !! FTNA2 !! BFR_E !! BFR_E !! BFR_E
| |
| - | |-
| |
| - | | Dilution || - || 1x || 3x || 5x||1x|| 3x || 5x||1x || 3x || 5x ||1x || 3x || 5x
| |
| - | |-
| |
| - | | Vector DNA || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1 || 1,1
| |
| - | |-
| |
| - | | Insert DNA || 0 || 0,4 || 1,6 || 2,0 || 0,10 || 0,47 || 0,79 || 0,18 || 0,52 || 0,90 || 0,40 || 1,16 || 2,0
| |
| - | |-
| |
| - | | Buffer || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1
| |
| - | |-
| |
| - | | T4 DNA-Ligase || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25 || 0,25
| |
| - | |-
| |
| - | | H<sub>2</sub>O || 7,65 || 7,25 || 6,5 || 5,65 || 7,5 || 7,20 || 6,85 || 7,50 || 7,20 || 6,85 || 7,25 || 6,50 || 5,65
| |
| - | |-
| |
| - | |}
| |
| - | ===Transformation===
| |
| - | --> Transformation of 5µl Sample into DH5α-Cells
| |
| - | --> Incubation o/n at 37°C after streating out on LB+amp plates using sterile beads
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! BFR !! FTNA1 !! FTNA2
| |
| - | |-
| |
| - | | 5,2ng || 5,51ng || 5,43ng
| |
| - | |-
| |
| - | | 15,6ng || 16,5ng || 16,27ng
| |
| - | |-
| |
| - | | 26,0ng || 27,4ng || 27,13ng
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/07 Monday - Cloning Results </h2>
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Sample !! CFU
| |
| - | |-
| |
| - | | Control || 13
| |
| - | |-
| |
| - | | tBFR 1 || 121
| |
| - | |-
| |
| - | | tBFR 3 || 38
| |
| - | |-
| |
| - | | tBFR 5 || 664
| |
| - | |-
| |
| - | | BFR 1 || 87
| |
| - | |-
| |
| - | | BFR 3 || 151
| |
| - | |-
| |
| - | | BFR 5 || 150
| |
| - | |-
| |
| - | | FTNA1 1 || 65
| |
| - | |-
| |
| - | | FTNA1 3 || 131
| |
| - | |-
| |
| - | | FTNA1 6 || 191
| |
| - | |-
| |
| - | | FTNA2 1 || 84
| |
| - | |-
| |
| - | | FTNA2 3 || 114
| |
| - | |-
| |
| - | | FTNA2 5 || 151
| |
| - | |}
| |
| - | --> picked 5 colos per construct for coloPCR (50µl TE-buffer; 96°C; 10min)
| |
| - | --> rescue plate
| |
| - |
| |
| - | '''colPCR Programm'''
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! 1x !! Mastermix 23x
| |
| - | |-
| |
| - | | nuc.free H<sub>2</sub>O || 13,7µl || 315,1µl
| |
| - | |-
| |
| - | | 10x Dream Taq Buffer || 2µl || 46µl
| |
| - | |-
| |
| - | | 25mM MgCl<sub>2</sub> || 1,2µl || 27,6µl
| |
| - | |-
| |
| - | | 10mM dNTPs || 0,5µl || 11,5µl
| |
| - | |-
| |
| - | | Primer T5 prom (PB16) || 0,5µl || 11,5µl
| |
| - | |-
| |
| - | | Primer T5 term (PB17) || 0,5µl || 11,5µl
| |
| - | |-
| |
| - | |
| |
| - | |-
| |
| - | | boiled colos || 1µl + 18,4ml Mastermix
| |
| - | |-
| |
| - | | Taq Polymerase || 0,5µl
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Temperature !! Time
| |
| - | |-
| |
| - | | 95°C || 3'
| |
| - | |-
| |
| - | |30 cycles
| |
| - | |-
| |
| - | | 95°C || 30''
| |
| - | |-
| |
| - | | 55°C || 30''
| |
| - | |-
| |
| - | | 72°C || 1'
| |
| - | |-
| |
| - | |
| |
| - | |-
| |
| - | | 72°C || 10'
| |
| - | |-
| |
| - | | 12°C || hold
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/08 Tuesday - Amplification of ferritin genes </h2>
| |
| - |
| |
| - | Primer designed for amplification:
| |
| - | <br/>
| |
| - | '''primer Bfr:'''
| |
| - |
| |
| - | FW: 5' GC G| G A T C C AAAGGTGATACTAAAGTTATAAATTATCTC(GC 23,33% Tm = 57,5) 3' OK
| |
| - |
| |
| - | RW: 5' GC A| A G C T T TCAACCTTCTTCGCG(GC 50% Tm = 58,5) 3' OK
| |
| - |
| |
| - | '''primer FtnA1:'''
| |
| - |
| |
| - | FW: 5' GC G| G A T C C GCAACCGCTGGAATG(GC 60% Tm = 60,5) 3' OK
| |
| - |
| |
| - | RW: 5' GC A| A G C T T TCAATGCAGCTGATGC(GC 50,0% Tm = 58,5) 3' OK
| |
| - |
| |
| - | '''primer FtnA2:'''
| |
| - |
| |
| - | FW: 5' GC G| G A T C C CTGAAACCAGAAATGATTG(GC 36,8% Tm = 65,1) 3' OK
| |
| - |
| |
| - | RW: 5' GC A| A G C T T TTAGTTTTGTGTGTCGAGG(GC 42,1% Tm = 56,8) 3' OK
| |
| - |
| |
| - |
| |
| - | Using the Thermoscientific PCR mastermix phusion polymerase, 2x 50 µl reactions per amplification:
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | Nuc-free H<sub>2</sub>O || 39,5 µl
| |
| - | |-
| |
| - | | 2x mastermix || 50,0 µl
| |
| - | |-
| |
| - | | FW primer || 5,0 µl
| |
| - | |-
| |
| - | | RW primer || 5,0 µl
| |
| - | |-
| |
| - | | template DNA || 0,5 µl
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | '''PCR programs:'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | || 98°C || 10s || 1x
| |
| - | |-
| |
| - | | || 98°C || 5s || 30x
| |
| - | |-
| |
| - | |primer|| Bfr/ FtnA1/ FtnA2 || 60,5°C/ 58,9°C/ 56,1°C || 30x
| |
| - | |-
| |
| - | | || 72°C || 10s || 30x
| |
| - | |-
| |
| - | | 72°C || 60s || Beispiel || 1x
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | <strong>Transformation of pQE_80L into DH5alpha</strong><br/>
| |
| - |
| |
| - | Plasmid from plasmid database of TU workgroup by Prof. Budisa was taken: <br/>
| |
| - | plasmid ID: 4; concentration = 308 ng/µl<br/>
| |
| - |
| |
| - | Unthaw 50 µl aliquot of DH5alpha or BL21 DE3 gold on ice for 10 min.Than Use 308 ng and 616 ng of plasmids to bind on Ca<big>2+</big>-surface of the cell membranes. Heat shock was performed for 30-90 s. The cells were incubated on ice for 2 min and than there was added 950 µl LB media. The cells were incubated at 37°C for 60 min. After this 70 µl of transformed E. coli suspension was streacked out onto a plate with the appropriate selection marker. The plate was incubated over night at 37°C. To prepare preculture 2 colonies were picked and added into 5 ml LB media + 5 µl AMP. The precultures were incubated over night at 37°C and 200 rpm.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/10 Thursday - Expression of Ferritin in LB </h2>
| |
| - |
| |
| - | →Innoculate 20ml LB + amp with 1ml of a preculture<br />
| |
| - |
| |
| - | {|
| |
| - | |-
| |
| - | ! Title !! No. !! Wavelength !! Absobance !! Volume
| |
| - | |-
| |
| - | | BfR || 1 || 600nm || 0,529A || 1,51ml
| |
| - | |-
| |
| - | | FTNA1|| 2 || 600nm || 0,307A || 3,25ml
| |
| - | |-
| |
| - | | FTNA2 || 3 || 600nm || 0,605A || 1,32ml
| |
| - | |}
| |
| - | →Incubation for 2h until OD<sub>600</sub>=0,6-08<br />
| |
| - | {|
| |
| - | |-
| |
| - | ! Title !! No. !! Wavelength !! Absobance
| |
| - | |-
| |
| - | | BfR || 1 || 600nm || 0,825A
| |
| - | |-
| |
| - | | FTNA1|| 2 || 600nm || 0,883A
| |
| - | |-
| |
| - | | FTNA2 || 3 || 600nm || 0,859A
| |
| - | |}
| |
| - | → Take SDS Sample non-induced<br />
| |
| - | →Induce with 20µl IPTG<br />
| |
| - | →Add Fe<sup>2+</sup><br />
| |
| - | →Expression: 4h at 22°C<br />
| |
| - | Cultivation was aborted because of heat development
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project"> .
| |
| - | <h2 class="sub-content-project-headline"> 04/14 Monday - Miniprep </h2>
| |
| - | Miniprep of the cell-seperation streak out of the cloning procedure from december 2013<br />
| |
| - | '''Miniprep-Kit of ThermoScientific'''<br />
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! Title !! Concentration in ng/µl
| |
| - | |-
| |
| - | | A4 || BfR || 66
| |
| - | |-
| |
| - | | B3 || FTNA1 || 138
| |
| - | |-
| |
| - | | C5 || FTNA2 || 88
| |
| - | |-
| |
| - | | D2 || BfR_Electro || 144
| |
| - | |}
| |
| - |
| |
| - | ==<big>'''DNA Concentration Determination'''</big>==
| |
| - | {|
| |
| - | |-
| |
| - | | Dilution Factor || 20
| |
| - | |-
| |
| - | | Integration Time || 1s
| |
| - | |-
| |
| - | | Factor || 50
| |
| - | |-
| |
| - | | Units || µg/ml
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! Title !! No. !! 260nm !! 280nm !! 320nm !! Ratio !! Conc.
| |
| - | |-
| |
| - | | A4 || BfR || 1 || 0,056 || -0,022|| -0,010 || 2,05 || 66
| |
| - | |-
| |
| - | | B3 || FTNA1 || 2 || 0,135 || 0,067 || -0,003 || 1,96 || 138
| |
| - | |-
| |
| - | | C5 || FTNA2 || 3 || 0,078 || 0,035|| -0,008 || 2,00 || 86
| |
| - | |-
| |
| - | | D2 || BfR-Electro || 4 || 0,150 || 0,078 || 0,006 || 2,02 || 144
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/16 Wednesday - Sequencing </h2>
| |
| - | Preparation of the sequencing for the Samples from 16.01.2013<br/>
| |
| - | {|
| |
| - | |-
| |
| - | | T5 prom. Primer || 3µl
| |
| - | |-
| |
| - | | Plasmid || 12µl
| |
| - | |}
| |
| - | {|
| |
| - | |-
| |
| - | | A4 || 6µl
| |
| - | |-
| |
| - | | B3 || 6µl
| |
| - | |-
| |
| - | | C5 || 6µl
| |
| - | |-
| |
| - | | D2 || 6µl
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/17 Thursday - Expression of BfR and FTNA1/2 </h2>
| |
| - | 4x sterile 250ml Erlenmeyer<br />
| |
| - | +50ml LB<br />
| |
| - | +50µl amp<br />
| |
| - | +2,5ml preculture<br />
| |
| - | Incubation: 1h at 30°C
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! No. !! Title !! Wavelength !! Absorbance
| |
| - | |-
| |
| - | | 1 || BfR || 600nm || 0,909A
| |
| - | |-
| |
| - | | 2 || FTNA1 || 600nm || 0,980A
| |
| - | |-
| |
| - | | 3 || FTNA2 || 600nm || 0,930A
| |
| - | |-
| |
| - | | 4 || BfR+20?? || 600nm || 0,970A
| |
| - | |}
| |
| - | →Take SDS-Sample<br />
| |
| - | →Induction with 50µl IPTG<br />
| |
| - | →Addition of 0,0525g Fe-citrate (20mM dissolved in 1ml sterile Water)<br />
| |
| - | →Incubation for 3h at 30°C<br />
| |
| - | →continue incubation o/n<br />
| |
| - | →Centrifuge with 6800g for 5min<br />
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/18 Friday - Production of LB-Plates </h2>
| |
| - |
| |
| - | ===Production of a Mutaflor-Supression-culture===
| |
| - | →10ml sterile LB<br />
| |
| - | →Add the content of a Mutaflor capsula (white pouder)<br />
| |
| - | Incubation: 37°C at 220rpm<br />
| |
| - | ===Production of Dilutions of the Supression-culture and out streaking===
| |
| - | →1µl of the supression-culture to 999µl LB (1:1000)<br />
| |
| - | →100µl of that dilution to 900µl LB (1:10<sup>4</sup>)<br />
| |
| - | →10µl of that dilution to 990µl LB (1:10<sup>5</sup>)<br />
| |
| - | →Streak out 50µl of each culture on LB with sterile beads<br />
| |
| - | Incubation: 37°C
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/24 Thursday - Mini-Prep </h2>
| |
| - | (Ammar Al-Shameri)
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | 1 || PSB || D40 || 9fP || CR/LB
| |
| - | |-
| |
| - | | 2 || PSB || D20 || 9fP || CR/LB
| |
| - | |-
| |
| - | | 3 || PSB || D40 || fs || LB/CR
| |
| - | |-
| |
| - | | 4 || PSB || D40 || LB/CR
| |
| - | |-
| |
| - | | 5 || PSB || AB || Turyu || CR/LB
| |
| - | |-
| |
| - | | 6 || PSB || D48 || 9fP || CB/CR
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/28 Monday -PCR Plasmid/ Primer and Parameter Check </h2>
| |
| - | (Salah and Christina)<br />
| |
| - |
| |
| - | Check of:<br />
| |
| - | -Plasmids/Primer<br />
| |
| - | -Annealing temperature<br />
| |
| - | -Phusion and Q5 Polymerase
| |
| - |
| |
| - | ===Plasmids===
| |
| - |
| |
| - | iGEM Plasmid pkD4 (Stock 200ng/µl) =>1:200 diluted = 1ng/µl
| |
| - |
| |
| - | WH (Willi Hauke) Plasmid pkD4 (0,72ng/µl)
| |
| - |
| |
| - | ===Primer===
| |
| - |
| |
| - | P1 FieF (iGEM) fwd tnaA KO (WH)
| |
| - |
| |
| - | P2 FieF (iGEM) rev tnaA KO (WH)
| |
| - |
| |
| - |
| |
| - | ===Combinations===
| |
| - |
| |
| - | (4 combinations with Phusion; 4 combinations with Q5 )
| |
| - |
| |
| - |
| |
| - | {| border="2"
| |
| - | |
| |
| - | | '''Plasmid'''
| |
| - | | '''Primer'''
| |
| - | | '''Primer'''
| |
| - | |-
| |
| - | | '''1. '''
| |
| - | | iGEM Plasmid
| |
| - | | P1 (iGEM)
| |
| - | | P2 (iGEM)
| |
| - | |-
| |
| - | | '''2. '''
| |
| - | | WH Plasmid
| |
| - | | fwd tnaA KO
| |
| - | | rev tnaA KO '''POSITIVCONTROL'''
| |
| - | |-
| |
| - | | '''3. '''
| |
| - | | iGEM Plasmid
| |
| - | | fwd tnaA KO
| |
| - | | rev tnaA KO
| |
| - | |-
| |
| - | | '''4. '''
| |
| - | | WH Plasmid
| |
| - | | P1 (iGEM)
| |
| - | | P2 (iGEM)
| |
| - | |}
| |
| - |
| |
| - | Each of the four combinations was prepared 3 times => for Gradient PCR
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Phusion Polymerase PCR Assay===
| |
| - |
| |
| - | 20µl Reaction
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! !! Plasmid !! Primer !! Primer
| |
| - | |-
| |
| - | | 1. || iGEM Plasmid || P1 (iGEM) || P2 (iGEM)
| |
| - | |-
| |
| - | | 2. || WH Plasmid || fwd tnaA KO || rev tnaA KO POSITIVCONTROL
| |
| - | |-
| |
| - | | 3. || iGEM Plasmid || fwd tnaA KO || rev tnaA KO
| |
| - | |-
| |
| - | | 4. || WH Plasmid || P1 (iGEM) || P2 (iGEM)
| |
| - | |-
| |
| - |
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Q5 Polymerase PCR Assay===
| |
| - |
| |
| - | 20µl Reaction
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''5xQ5 Reaction Puffer (G2 runder Behälter)'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''4µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''1x'''
| |
| - | |-
| |
| - | | 10mM dNTPs ||0,4µl||200µM
| |
| - | |-
| |
| - | | 10µM forward Primer||1µl||0,5µM
| |
| - | |-
| |
| - | | 10µM reverse Primer||1µl||0,5µM
| |
| - | |-
| |
| - | | Template DNA||1µl||1pg-1ng sein
| |
| - | |-
| |
| - | | Q5 High Fidelity DNA Polymerase||0,2µl||0,02U/µl
| |
| - | |-
| |
| - | | GC enhancer||4 µl||
| |
| - | |-
| |
| - | | Nuclease-Free Water ||to 20µl||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | ||8,4µl||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | ===FUR PCR Assay===
| |
| - | <br/>
| |
| - | (Along with the above mentioned PCR Assays another Gradient PCR with FUR-Primer is performed)<br/>
| |
| - |
| |
| - | Aliquot of the FUR Primer (100µM) 1:10 diluted =>10µM<br/>
| |
| - |
| |
| - | 20µl Reaction with Phusion-Polymerase (see above)<br/>
| |
| - |
| |
| - | with WH Plasmid and P1 FUR & P2 FUR Primer<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Gradient PCR Program===
| |
| - |
| |
| - | Thermocycling Conditions for the Gradient PCR <br/>
| |
| - |
| |
| - | Programm-NAME: Q5 grad trp KO<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Step'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''TEMP'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''TIME'''
| |
| - | |-
| |
| - | | Initial Denaturation||98°C||30 sec
| |
| - | |-
| |
| - | | 5 Cycles||98°C||8 sec
| |
| - | |-
| |
| - | | ||64±5°C||25 sec GRADIENT
| |
| - | |-
| |
| - | | ||72°C||Trp ko: 1528bpà45s
| |
| - | |-
| |
| - | | 25 Cycles||98°C||8 sec
| |
| - | |-
| |
| - | | ||72°C||70 sec
| |
| - | |-
| |
| - | | Final Extension||72°C||2 min
| |
| - | |-
| |
| - | | Hold||8°C||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ===Gel-Electrophoresis===
| |
| - |
| |
| - | 1% Agarose Gel<br/>
| |
| - |
| |
| - | 5µl Sample +1µl loading dye (1:6)<br/>
| |
| - |
| |
| - | 8µl diluted GeneRuler Mix ladder<br/>
| |
| - |
| |
| - | MODE: 90V 25min<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | '''Sample label:'''
| |
| - |
| |
| - |
| |
| - |
| |
| - | P = with Phusion Polymerase<br/>
| |
| - |
| |
| - | Q = with Q5 Polymerase<br/>
| |
| - |
| |
| - | 1 = 1. Kombination<br/>
| |
| - |
| |
| - | 2 = 2. Kombination<br/>
| |
| - |
| |
| - | 3 = 3. Kombination<br/>
| |
| - |
| |
| - | 4 = 4. Kombination<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | '''Gradient'''
| |
| - |
| |
| - | 1 = 59°C
| |
| - |
| |
| - | 4 = 63°C
| |
| - |
| |
| - | 8 = 69°C
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Gel 1 Phusion'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | P FUR||P1||P1||P1||P2||P2||P2||Marker||P3||P3||P3||P4||P4||P4
| |
| - | |-
| |
| - | | ||||||||||||||||||||||||||
| |
| - | |-
| |
| - | | 8||1||4||8||1||4||8||||1||4||8||1||4||8
| |
| - | |-
| |
| - | | Gel 2 Q5||||||||||||||||||||||||||
| |
| - | |-
| |
| - | | ||Q1||Q1||Q1||Q2||Q2||Q2||Marker||Q3||Q3||Q3||Q4||Q4||Q4
| |
| - | |-
| |
| - | | ||||||||||||||||||||||||||
| |
| - | |-
| |
| - | | ||1||4||8||1||4||8||||1||4||8||1||4||8
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | ====Results====
| |
| - |
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Gel 1 Phusion Result:'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''For FUR-Primer no Amplifikation'''
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||For iGEM-Plasmid no Amplifikation
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||Combination 2 (WH Plasmid & Primer)= only non-specific amplification
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||Combination 4 (WH Plasmid & iGEM Primer)=Bands for 59°C and 63°C + non-specific amplification
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | align="center" style="background:#f0f0f0;"|'''Gel 2 Q5 Result:'''||For iGEM-Plasmid no Amplifikation
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||Combination 2 (WH Plasmid & Primer) = bands for all three annealing temperature observed+non-specific amplification
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||Combination 4 (WH Plasmid & iGEM Primer)= Bands at all three annealing temperature+non-specific amplification
| |
| - | |-
| |
| - | |
| |
| - | |}<br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/2/2c/Team_berlin_0001.jpg" alt="" />
| |
| - | <br />
| |
| - | iGEM Plasmid not to be used anymore.
| |
| - | FUR need to be checked again.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04/29 Tuesday - Mini-Prep </h2>
| |
| - | (Aritra)<br />
| |
| - | [pKD46 + DH5α]→ 155ng/µl<br />
| |
| - | [pKD4 + DH5α]→ 302ng/µl<br />
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/02 Friday - Genomic DNA extraction from Psedomonas putida </h2>
| |
| - | (Johann)<br />
| |
| - |
| |
| - | -->see Protocol Wizard ® Genomic DNA Purification Kit
| |
| - |
| |
| - | - Strain from VLB Martin Senz PHO Psedomonas putida KT2242 B_0712 from 29.04.2014 culture<br/>
| |
| - | - 2 days in 30°C Shaking Incubator.<br/>
| |
| - | - Mikro 22R Centrifuge Hettich (16000g)<br/>
| |
| - |
| |
| - | Final DNA Concentration measured after purification = 95ng/µl
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 02.05.2014 - DNA Digestion to test Plasmids </h2>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Samples'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Conc. DNA (ng/µl)'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Enzymes used'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Expected band length'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Evaluation'''
| |
| - | |-
| |
| - | | pKD4||302||Xbal||1874,1393||Possible plasmid, failed
| |
| - | |-
| |
| - | | pKD46||40||EcoRI||4820,1509||Failed
| |
| - | |-
| |
| - | | pKD46||39||EcoRI||4820,1509||Failed
| |
| - | |-
| |
| - | | pKD4||60||Xbal||1874,1393||Possible(passed); whole plasmid
| |
| - | |-
| |
| - | | pSBAC3||88||SpeI/EcoRI||2047,22||Possible (passed); smallpattern
| |
| - | |-
| |
| - | | pSBAC3-D20-GFP||115||SpeI/EcoRI||2047,957||whole plasmid; failed
| |
| - | |-
| |
| - | | pSBAC3-D40-Bfr||86||SpeI/EcoRI||2047, 774||Whole plasmid; failed
| |
| - | |-
| |
| - | | pSBAC3-D40-GFP||80||SpeI/EcoRI||2047,1014||Whole plasmid; failed
| |
| - | |-
| |
| - | | pSBAC3-AB-Tyrosin||110||SpeI/EcoRI||2047,3319||Failed
| |
| - | |-
| |
| - | | pSBAC3-D40||164||SpeI/EcoRI||2047,267||Failed
| |
| - | |-
| |
| - | | pSBAC3-D40-GFP-ssr||106||SpeI/EcoRI||2047,1032||Passed; whole plasmid
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - | ==Gel Electrophoresis==
| |
| - | '''Key:'''<br>
| |
| - | -band1: Ladder<br />
| |
| - | -band5:Ladder<br />
| |
| - | -residual bands, see table above
| |
| - | <br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/5/5b/Team_berlin_0002.png" alt="" /><br/>
| |
| - | ===Results===
| |
| - |
| |
| - | pKD4 with 60(ng/µl) best candidate for PCR.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/05 Monday - Preculture of strains from Budisa strain database in LB Medium </h2>
| |
| - | (Johann)
| |
| - |
| |
| - | 1. BU31 (pKD4) in 50 ml LB+Kanamycin+Ampicillin 37°C 220 rpm<br/>
| |
| - | 2. BU32 (pKD46) in 50 ml LB+Ampicillin 30°C 240rpm<br/>
| |
| - | 3. BU31 (pKD46) in LB plate+Ampicillin 30°C <br/>
| |
| - |
| |
| - | ==Midiprep==
| |
| - | (christina)
| |
| - |
| |
| - | Midiprep of BU31 (pKD4) and BU32 (pKD46) from strain Database.<br/>
| |
| - |
| |
| - | Concentration pKD4 = 104 ng/µl<br/>
| |
| - |
| |
| - | Concentartion pKD46 = 161 ng/µl<br/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/06 Tuesday - Gel-Electrophoresis </h2>
| |
| - | (Aritra)
| |
| - | Kanamycin Cassette PCR (pKD4 from MidiPrep 05.05.2014 104ng/µl Christina)<br />
| |
| - |
| |
| - | '''Key:'''<br />
| |
| - | - 1st: Ladder<br />
| |
| - |
| |
| - | - 2nd and 3rd: Fief<br />
| |
| - |
| |
| - | - 4th: Negative control<br />
| |
| - | - 7th and 8th: FUR<br />
| |
| - | <img src="https://static.igem.org/mediawiki/2014/c/c3/Team_berlin_0003.jpg" alt="" />
| |
| - | <br/>
| |
| - | ===Results===
| |
| - | FieF worked well; FUR no bands
| |
| - |
| |
| - |
| |
| - | ==Restriction digest of Plasmid pKD4 and pKD46==
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | ||pKD4||pKD46
| |
| - | |-
| |
| - | | Plamid ||19.2µl||12.5µl
| |
| - | |-
| |
| - | | Digestion Enzym||Xbal 1µl||Xmal 1µl
| |
| - | |-
| |
| - | | nuclease free H2O||24.8µl||31.5µl
| |
| - | |-
| |
| - | | 10xFO green Buffer||5µl||5µl
| |
| - | |-
| |
| - | | Total||50µl||50µl
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - | - Both the reaction mix incubated at 37°C for 1 hr.
| |
| - | - After that another 1 hr at RT.
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Gel-Electrophoresis===
| |
| - | - 20µl of both the samples together with whole plasmids loaded on 1% Agarose Gel<br />
| |
| - | ====Results====
| |
| - | '''Key:'''<br />
| |
| - | -1st: pKD4 (XbaI digested)<br />
| |
| - | -2nd: pKD4 (undigested)<br />
| |
| - | - 3rd: Ladder<br />
| |
| - | - 4th: pKD46 (undigested)<br />
| |
| - | -5th: pKD46 (XmaI digested)<br />
| |
| - |
| |
| - |
| |
| - | ===Gel Extraction===
| |
| - | -Used GeneJet GelExtraction Kit by Thermo Scientific
| |
| - | ====Results====
| |
| - | DNA Concentration: 16ng/µl
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/16 Friday - Biotransformation of Ferritin and pKD46 in Nissle and DH5-α strain </h2>
| |
| - | concentration Ferritin 120ng/µl<br>
| |
| - | concentration pKD 46 95 ng/µl<br/>
| |
| - |
| |
| - | Protocol
| |
| - | *electro competent cells of DH5α and Nissle were thawed on ice
| |
| - | *0.4µl of Ferritin and 0.53 µl of pKD46 was added to the cells
| |
| - | *cells with DNA were transferred to cuvettes for electroporation, then 950ml (LB-medium) was added to the cells immediately
| |
| - | *incubation at 37°C for ferritin, 30°C for pKd46 for 1 h with shaking
| |
| - | *the cells were plated on Amp plates for pKD46 and CM for Ferritin
| |
| - | *Transformation did not work.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/21 Wednesday - PCR Gel-Extraction of FieF </h2>
| |
| - | (Christina)<br />
| |
| - |
| |
| - | 1% Agarosegel + 0,5µl EtBr<br />
| |
| - | 45µl Sample+9µl Loading Dye<br />
| |
| - | 8µl Ladder<br />
| |
| - | --> Gel-Extraction Kit used with 50µl nuclease-free water for elution<br />
| |
| - | [[Datei:140521 DNA conc.jpg|miniatur|zentriert]]
| |
| - | ==Gene Knockout of FieF==
| |
| - | (Salah)
| |
| - | ===Preparation of the preculture===
| |
| - | '''Used Precultures:'''<br />
| |
| - | - RV +pKD46<br />
| |
| - | - WM10 +pKD46<br />
| |
| - | The precultures was diluted to OD<sub>600</sub>=0,1 and then incubated for 2h at 30°C until they reach OD<sub>600</sub>=0,6<br />
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Title !! Wavelength !! Absorbance
| |
| - | |-
| |
| - | | Reference || 600nm || 0,000A
| |
| - | |-
| |
| - | | RV+pKD46 || 600nm || 0,066A
| |
| - | |-
| |
| - | | WM10+pKD46 || 600nm || 0,050A
| |
| - | |}
| |
| - | (The culture was diluted 1/10 for the OD measurement)<br />
| |
| - | →The precultures are ready for the Rekombinase induction with Arabinose
| |
| - | ===Induction with Arabinose===
| |
| - | The cultures have been induced with 500µl arabinose (1M)
| |
| - | →After the induction, the cultures have to be incubated for further 30min at 30°C
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Title !! Wavelength !! Absorbance
| |
| - | |-
| |
| - | | Reference || 600nm || 0,000A
| |
| - | |-
| |
| - | | RV+pKD46 || 600nm || 0,084A
| |
| - | |-
| |
| - | | WM10+pKD46 || 600nm || 0,067A
| |
| - | |}
| |
| - | →From now on the cultures need to be cooled on ice
| |
| - | ===Preparation for the Electroporation===
| |
| - |
| |
| - | ===Electroporation===
| |
| - | '''Electroporation-Program: Ec1'''<br />
| |
| - | -Amount of DNA sample: 75-150ng<br />
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Sample !! Conc !! Used Volume !! Strain !! Electroporation Time
| |
| - | |-
| |
| - | | FieF1 || 42ng/µl || 2µl (84ng) || RV || 4,4ms
| |
| - | |-
| |
| - | | FieF2 || 9ng/µl || 10µl (90ng) || RV || 4,1ms
| |
| - | |-
| |
| - | | FieF1 || 42ng/µl || 2µl (84ng) || WM10 || 4,3ms
| |
| - | |-
| |
| - | | FieF2 || 9ng/µl || 10µl (90ng) || WM10 || 4,8ms
| |
| - | |}
| |
| - | After the transformation, the cultures was immediately prepared with 950µl SOC and incubated for 60min at 30°C without antibiotics --> recovery
| |
| - | ===Selection-cultures===
| |
| - | The selection of positive clones is done by using LB plates with kan+amp<br />
| |
| - | Incubation: 30°C o/n
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/22 Thursday - Transformation of pKD46 </h2>
| |
| - | (Salah)
| |
| - | ===Preparation of the preculture===
| |
| - | '''Used Precultures:'''<br />
| |
| - | - MG1655<br />
| |
| - | - Nissle<br />
| |
| - | The precultures was diluted to OD<sub>600</sub>=0,1 and then incubated for 2h at 30°C until they reach OD<sub>600</sub>=0,6<br />
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Title !! Wavelength !! Absorbance
| |
| - | |-
| |
| - | | Reference || 600nm || 0,000A
| |
| - | |-
| |
| - | | MG1655 || 600nm || 0,062A
| |
| - | |-
| |
| - | | NIssle || 600nm || 0,054A
| |
| - | |}
| |
| - | (The culture was diluted 1/10 for the OD measurement)<br />
| |
| - |
| |
| - | →From now on the cultures need to be cooled on ice
| |
| - | ===Preparation for the Electroporation===
| |
| - |
| |
| - | ===Electroporation===
| |
| - | '''Electroporation-Program: Ec1'''<br />
| |
| - | -Amount of DNA sample: 75-150ng<br />
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Sample !! Conc !! Used Volume !! Strain !! Electroporation Time
| |
| - | |-
| |
| - | | MG1655 1 || 42ng/µl || 2µl (84ng) || RV || 5,4ms
| |
| - | |-
| |
| - | | MG1655 2 || 9ng/µl || 10µl (90ng) || RV || 5,8ms
| |
| - | |-
| |
| - | | Nissle 1 || 42ng/µl || 2µl (84ng) || WM10 || 0,7ms (thrown away)
| |
| - | |-
| |
| - | | Nissle 2 || 9ng/µl || 10µl (90ng) || WM10 || 5,8ms
| |
| - | |}
| |
| - | After the transformation, the cultures was immediately prepared with 950µl SOC and incubated for 60min at 30°C without antibiotics --> recovery
| |
| - | ===Selection-cultures===
| |
| - | The selection of positive clones is done by using LB plates with amp<br />
| |
| - | Incubation: 30°C o/n
| |
| - |
| |
| - | == PCR of ATPCS and PPMT ==
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Mastermix !! ATPCS !! PPMT
| |
| - | |-
| |
| - | | nuc.free water|| 18.9 || 17.5
| |
| - | |-
| |
| - | | 2x Fusion Mastermix || 25 || 25
| |
| - | |-
| |
| - | | Fw Primar || 2.5 || 2.5
| |
| - | |-
| |
| - | | Rw Primar || 2.5|| 2.5
| |
| - | |-
| |
| - | | template DNA || 1.1 || 2.5
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! ATPCS !! Programm
| |
| - | |-
| |
| - | | 98°C || 10
| |
| - | |-
| |
| - | | 98°C|| 5 30 cycles
| |
| - | |-
| |
| - | | 62°C|| 5 30 cycles
| |
| - | |-
| |
| - | | 72°C || 5 30 cycles
| |
| - | |-
| |
| - | | 72°C|| 60 secs
| |
| - | |-
| |
| - | | 8°C|| store
| |
| - | |}
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PPMT!! Programm
| |
| - | |-
| |
| - | | 98°C || 10
| |
| - | |-
| |
| - | | 98°C|| 5 30 cycles
| |
| - | |-
| |
| - | | 63,1°C|| 5 30 cycles
| |
| - | |-
| |
| - | | 72°C || 21 30 cycles
| |
| - | |-
| |
| - | | 72°C|| 60 secs
| |
| - | |-
| |
| - | | 8°C|| store
| |
| - | |}
| |
| - |
| |
| - | ATPCS 1469 bp; PPMT 234 bp
| |
| - | --> wrong Programm
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/23 Friday - Digestion of Vector for Cloning </h2>
| |
| - | Plasmid: pQE80L<br />
| |
| - |
| |
| - | Old Plasmid of Johann: <br />
| |
| - | -P1≈1000ng<br/>
| |
| - | -P3≈400ng<br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! P3 !! !! P1 !!
| |
| - | |-
| |
| - | | SacI || 1µl || SacI || 1µl
| |
| - | |-
| |
| - | | HindIII || 1µl || BamHI || 1µl
| |
| - | |-
| |
| - | | Buffer || 2µl || Buffer || 2µl
| |
| - | |-
| |
| - | | Plasmid 1 || 4µl || Plasmid 3 || 16µl
| |
| - | |-
| |
| - | | nuc.water || 12µl || nuc. water || 0µl
| |
| - | |}
| |
| - | --> Incubation: 1,5h at 37°C<br />
| |
| - | --> Deactivation: 20min at 80°C
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/27 Tuesday - Production of LB Plates </h2>
| |
| - | (Salah and Christina)<br/>
| |
| - |
| |
| - | LB Agar Plates prepared:<br/>
| |
| - |
| |
| - | 4x with Ampicillin (25µl in 25ml)<br/>
| |
| - |
| |
| - | 4x with Ampicillin+ Kanamycin (25µl+25µl in 25ml)<br/>
| |
| - |
| |
| - | 4x Cm (25µl in 25ml)
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05/28 Wednesday </h2>
| |
| - | Picked up from MPI: AMB-1 and Microfluidic Chip. (From the two AMB-1 cultures, one is in the freeze and the other one in the RT-Incubator)<br/>
| |
| - |
| |
| - | Digestion of pQE80L: <br/>
| |
| - |
| |
| - | Marker: 2 log DNA ladder 5µl<br/>
| |
| - |
| |
| - | Sample 1: P1 Sacl-BamHI 10µl<br/>
| |
| - |
| |
| - | Sample 2: P3 Sacl-HindIII 10µl<br/>
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/02 Monday - Colony PCR and analytical gel electrophoresis for identifying the right clones </h2>
| |
| - |
| |
| - | Primer: C1 C2<br/>
| |
| - |
| |
| - | C2 C5<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Components'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''20 µL Reaktion'''
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | ||
| |
| - | |-
| |
| - | | Nuc. Free water||13.7
| |
| - | |-
| |
| - | | 10x Dream Taq Polymerase Puffer||2
| |
| - | |-
| |
| - | | 25mM MgCl2||1.2
| |
| - | |-
| |
| - | | 10mM dNTPs||0.5
| |
| - | |-
| |
| - | | 10mM FW Primer||0.5
| |
| - | |-
| |
| - | | 10mM RW Primer||0.5
| |
| - | |-
| |
| - | | Colony||1
| |
| - | |-
| |
| - | | Taq DNA Pol||0.6
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Denaturation'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''95°C'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''10min'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | Denaturation||95°C||30sec||
| |
| - | |-
| |
| - | | Annealing||55°C||30 sec||
| |
| - | |-
| |
| - | | Extension||72°C||X min||30x
| |
| - | |-
| |
| - | | Extension||72°C||10 min||
| |
| - | |-
| |
| - | | End||12||hold||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Backup plates done. Cells given direct to PCR. 11 colonies selected.<br/>
| |
| - |
| |
| - | Colony PCR with some of the clones performed. (All negative)
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/03 Tuesday - Strategy 2: PCR of ATPCS 2PPMT </h2>
| |
| - | (Virginia and Aritra)<br />
| |
| - | ===PCR Assay===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! Sample1(PPMT) !! Sample2(ATPCS)
| |
| - | |-
| |
| - | | nuc. free water || 17,5µl || 18,9µl
| |
| - | |-
| |
| - | | FW Primer || 2,5µl || 2,5µl
| |
| - | |-
| |
| - | | RW Primer || 2,5µl || 2,5µl
| |
| - | |-
| |
| - | | 2x Phusion Mastermix || 25µl || 25µl
| |
| - | |-
| |
| - | | Template DNA || 2,5µl (250ng) || 1,1µl (250ng)
| |
| - | |}
| |
| - |
| |
| - | ===PCR Program===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PPMT (234bp) !! !! ATPCS (1469bp) !!
| |
| - | |-
| |
| - | | 98°C || 10' || 98°C || 10'
| |
| - | |-
| |
| - | |30cycles start
| |
| - | |-
| |
| - | | 98°C || 5" || 98°C || 5"
| |
| - | |-
| |
| - | | 62°C || 5" || 63°C || 5"
| |
| - | |-
| |
| - | | 72°C || 10" || 72°C || 45"
| |
| - | |-
| |
| - | |30cycles stop
| |
| - | |-
| |
| - | | 72°C || 1' || 72°C || 1'
| |
| - | |-
| |
| - | | 8°C || hold || 8°C || hold
| |
| - | |}
| |
| - | ===Gel-Electrophoresis===
| |
| - | '''Key:''' <br />
| |
| - | 2nd band= Ladder<br />
| |
| - | 3rd band= PPMT<br />
| |
| - | 5th band= ATPCS<br />
| |
| - |
| |
| - | <img src="https://static.igem.org/mediawiki/2014/c/c3/Team_berlin_0004.jpg" alt="" />
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/04 Wednesday - PCR amplification of ATPCS and PPMT </h2>
| |
| - |
| |
| - | (see under protocol "Amplification BamHI_PPMT_Sacl and Sacl_ATPCS_HindIII"
| |
| - |
| |
| - | After Gel: 1 Band visible for ATPCS around 1500bp (expected); no band for PPMT
| |
| - |
| |
| - | Purification of the ATPCS-PCR product from gel.
| |
| - |
| |
| - | conc. of ATPCS-fragment 6ng/microL
| |
| - |
| |
| - | freezed at -20°C on 1st floor
| |
| - | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_berlin_0005.jpg" alt="" />
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 04.06.2014 - Colony PCR for Knockout </h2>
| |
| - |
| |
| - | 6 clones were picked from 2 Agar plates; Agar plates labelled with numbers with RED
| |
| - |
| |
| - | 3 PCR: a) Primers C1, C3
| |
| - |
| |
| - | b) Primers C2, C4
| |
| - |
| |
| - | c) Primers C1, C5
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''BandNr:'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Clone Nr:'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''PCR type'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Primers'''
| |
| - | |-
| |
| - | | 1||6||C||C1,C5
| |
| - | |-
| |
| - | | 2||1||A||C1,C3
| |
| - | |-
| |
| - | | 3||2||A||C1, C3
| |
| - | |-
| |
| - | | 4||3||A||C1, C3
| |
| - | |-
| |
| - | | 5||4||A||C1, C3
| |
| - | |-
| |
| - | | 6||5||A||C1, C3
| |
| - | |-
| |
| - | | 7||6||A||C1, C3
| |
| - | |-
| |
| - | | 8||1||B||C2,C4
| |
| - | |-
| |
| - | | 9||2||B||C2,C4
| |
| - | |-
| |
| - | | 10||Marker||||
| |
| - | |-
| |
| - | | 11||3||B||C2,C4
| |
| - | |-
| |
| - | | 12||4||B||C2,C4
| |
| - | |-
| |
| - | | 13||5||B||C2,C4
| |
| - | |-
| |
| - | | 14||6||B||C2,C4
| |
| - | |-
| |
| - | | 15||1||C||C1,C5
| |
| - | |-
| |
| - | | 16||2||C||C2,C4
| |
| - | |-
| |
| - | | 17||3||C||C2,C4
| |
| - | |-
| |
| - | | 18||4||C||C2,C4
| |
| - | |-
| |
| - | | 19||5||C||C2,C4
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Results: all samples with same primers are identical
| |
| - |
| |
| - | PCR A: one band an dimer (900-800bp)
| |
| - |
| |
| - | PCR B: one band (1200bp)
| |
| - |
| |
| - | PCR C: two bands (800 and 400 bp)
| |
| - |
| |
| - | - 2 of the colonies are quite luckily mix colonies that contains our knockout, bands are shown for wildtype and 2 show amplification for kanR cassette
| |
| - | - they should be transfered to a new plate for single colony test
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/05 Thursday - New PCR protocol for ATPCS and PPMT with Q5 High Fidelity MasterMix </h2>
| |
| - |
| |
| - | '''PPMT PCR Reaction:'''
| |
| - |
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Component'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''25µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Final Conc.'''
| |
| - | |-
| |
| - | | 2x Q5 MM||12. Mai||1x
| |
| - | |-
| |
| - | | 10µM FW Primer||02. Mai||0,5µM
| |
| - | |-
| |
| - | | 10µM RW Primer||2,5||0,5µM
| |
| - | |-
| |
| - | | 95ng/µL Putida genomic DNA||2.63 (250ng)||9.99 ng/µL
| |
| - | |-
| |
| - | | Nuc. Free water||Apr 87||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | '''ATPCS PCR Reaktion
| |
| - |
| |
| - | result'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Component'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''25µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Final Conc.'''
| |
| - | |-
| |
| - | | 2x Q5 MM||1.,5||1x
| |
| - | |-
| |
| - | | 10µM FW Primer||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 10µM RW Primer||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 230ng/µL Arabidopsis cDNA||1.1 (250ng)||10 ng/µL
| |
| - | |-
| |
| - | | Nuc. Free water||08. Sep||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | PCR Program
| |
| - | '''PPMT 234 bp
| |
| - | result'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''denaturing'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''98°C'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''180sec'''
| |
| - | |-
| |
| - | | Denaturing||98°C||10 sec
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | Annealing 30x||||
| |
| - | |-
| |
| - | | ||64°C||30 sec
| |
| - | |-
| |
| - | | Elongation||72°C||20 sec
| |
| - | |-
| |
| - | | Final Elongation||72°C||2min
| |
| - | |-
| |
| - | | Store||8°C||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - | '''ATPCS 1469bp
| |
| - | result'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''denaturing'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''98°C'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''30 sec'''
| |
| - | |-
| |
| - | | Denaturing||98°C||10 sec
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | Annealing 30x||||
| |
| - | |-
| |
| - | | ||62°C||30 sec
| |
| - | |-
| |
| - | | Elongation||72°C||60 sec
| |
| - | |-
| |
| - | | Final Elongation||72°C||2min
| |
| - | |-
| |
| - | | Store||8°C||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 05.06.2014 </h2>
| |
| - |
| |
| - | Cryostocks: RV 308 ferritin (Cm)<br/>
| |
| - |
| |
| - | Nissle ferritin (Cm)<br/>
| |
| - |
| |
| - | 500 µl 7%DMSO+ 500 µl Cell Culture medium => -80°C <br/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/06 Friday - PCR for ATPCS and PPMT with Q5 High Fidelity MasterMix </h2>
| |
| - |
| |
| - | PPMT PCR Reaction:<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Component'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''25µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''25µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Final Conc.'''
| |
| - | |-
| |
| - | | 2x Q5 MM||12. Mai||12,5||1x
| |
| - | |-
| |
| - | | 10µM FW Primer||02. Mai||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 10µM RW Primer||2,5||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 95ng/µL Putida genomic DNA||5,26 (500ng)||5,26 (500 ng)||
| |
| - | |-
| |
| - | | Nuc. Free water||Apr 87||4,74||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | ATPCS PCR Reaction<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Component'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''25µl'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''Final Conc.'''
| |
| - | |-
| |
| - | | 2x Q5 MM||12. Mai||1x
| |
| - | |-
| |
| - | | 10µM FW Primer||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 10µM RW Primer||Jan 25||0,5µM
| |
| - | |-
| |
| - | | 230ng/µL Arabidopsis cDNA||2.2 (500ng)||
| |
| - | |-
| |
| - | | Nuc. Free water||08. Sep||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | PCR programm<br/>
| |
| - |
| |
| - | PPMT 234 bp<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''denaturing'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''98°C'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''120sec'''
| |
| - | |-
| |
| - | | Denaturing||98°C||10 sec
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | Annealing 30x||||
| |
| - | |-
| |
| - | | ||64°C||30 sec
| |
| - | |-
| |
| - | | Elongation||72°C||20 sec
| |
| - | |-
| |
| - | | Final Elongation||72°C||2min
| |
| - | |-
| |
| - | | Store||8°C||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - | ATPCS 1469bp
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''denaturing'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''98°C'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''30 sec'''
| |
| - | |-
| |
| - | | Denaturing||98°C||10 sec
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | Annealing 30x||||
| |
| - | |-
| |
| - | | ||62°C||30 sec
| |
| - | |-
| |
| - | | Elongation||72°C||60 sec
| |
| - | |-
| |
| - | | Final Elongation||72°C||2min
| |
| - | |-
| |
| - | | Store||8°C||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | 10µl of PCR reaction + 2µl 6XDNA loading dye was loaded onto a 1 % agarose gel<br/>
| |
| - |
| |
| - | ATPCS (one clear thick epxacted band at Test of purity and quality of genomic DNA by running it through a 1% agarose gel1400 bp)<br/>
| |
| - |
| |
| - | PPMT1 (no band)<br/>
| |
| - |
| |
| - | PPMT2 (no band)<br/>
| |
| - | <br/>>
| |
| - |
| |
| - |
| |
| - | '''Test of genomic DNA of P.Putida for purity and quality by running it on a 1% agarose gel'''
| |
| - | <br/>
| |
| - | - looks fine on a gel. One clear band over 10 000 bp<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | '''!!! Improve PPMT !!!:'''
| |
| - |
| |
| - | Try using DMSO (NEB PCR grad) and set Annealingtemperature down to 55°C<br/>
| |
| - |
| |
| - | Try different DMSO conc. : 0; 1; 5; 8 %
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/11 Wednesday - Continuation of work from 06.06.2014: PCR for (ATPCS and) PPMT with Q5 </h2>
| |
| - |
| |
| - |
| |
| - |
| |
| - | PPMT PCR without success last weak. Therefore the temperature of the annealing step should reduce from 64°C up to 55°C in the pcr programm.<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | '''1) corrected pcr programm for PPMT'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''T [°C]'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''t [s]'''
| |
| - | |-
| |
| - | | Denaturating||98||120
| |
| - | |-
| |
| - | | Denaturating||98||10
| |
| - | |-
| |
| - | | Annealing||55||30
| |
| - | |-
| |
| - | | Elongation||72||60
| |
| - | |-
| |
| - | | Final Elongation||72||120
| |
| - | |-
| |
| - | | Store||8||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | '''2) PPMT pcr reaction'''
| |
| - |
| |
| - | Different samples with concentrations on DMSO 0%, 1%, 5%, 8% were prepared. Use 99,97% DMSO the samples were prepared with the following pattern:
| |
| - | The final volume is 25μl.<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''#1 [μl]'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''#2 [μl]'''
| |
| - | |-
| |
| - | | Q5 MM||12,5||12,5
| |
| - | |-
| |
| - | | 10 μl FW Primer||2,5||1,25
| |
| - | |-
| |
| - | | 10 μl RW Primer||2,5||1,25
| |
| - | |-
| |
| - | | Putida genomic DNA 95 ng/μl|| 5,26|| 5,26
| |
| - | |-
| |
| - | | dazu 1.: DMSO,||0||0
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O||2,24||4,74
| |
| - | |-
| |
| - | | dazu 2.: DMSO,||0,25||0,25
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O||1,99||4,49
| |
| - | |-
| |
| - | | dazu 3.: DMSO,||1,25||1,25
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O||0,99||3,49
| |
| - | |-
| |
| - | | dazu 4.: DMSO,||2||2
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O||0,24||2,74
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | '''the 7 samples were labeled by the following schema:'''
| |
| - |
| |
| - | PPMT.1.1, PPMT.1.2, PPMT.1.3, PPMT.1.4 (Primer je 2,5μl, DMSO Konz. 0,1,5,8 %)
| |
| - |
| |
| - | PPMT.2.2, PPMT.2.3., PPMT.2.4 (Primer je 1,25μl, DMSO Konz. 1,5,8 %)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/12 Thursday - PCR of PPMT </h2>
| |
| - | Amplification of PPMT with DMSO<br />
| |
| - | ''(Continuation of work from 06.06.2014: PCR for PPMT with Q5''
| |
| - |
| |
| - | For Protocol please see Labjournal from 11.06.2014<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | '''following labeling for 8 samples was used:'''
| |
| - |
| |
| - | PPMT.1.1, PPMT.1.2, PPMT.1.3, PPMT.1.4 (Primer je 2,5μl, DMSO Konz. 0,1,5,8 %)
| |
| - |
| |
| - | PPMT 2.1, PPMT.2.2, PPMT.2.3., PPMT.2.4 (Primer je 1,25μl, DMSO Konz. 0,1,5,8 %)
| |
| - |
| |
| - |
| |
| - |
| |
| - | ===Gel Electrophoresis===
| |
| - |
| |
| - | 1µl loading buffer+5 µl sample loaded on 1% agarose gel; 90V<br />
| |
| - |
| |
| - | (rest of sample in iGEM freezer)
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | ====Results====
| |
| - |
| |
| - | contamination from water (band 3000bp for every sample)<br/>
| |
| - |
| |
| - | with higher DMSO conc. (5% and 8%) bands from visible
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/17 Tuesday - PCR Amplification of ATPCS from cDNA </h2>
| |
| - |
| |
| - | ===PCR-pipetting scheme===
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Volume [µl] !! Chemicals
| |
| - | |-
| |
| - | | 12,5 || 2x Q5 Mastermix
| |
| - | |-
| |
| - | | 1,25 || 10µM fwd Primer
| |
| - | |-
| |
| - | | 1,25 || 10µM rev Primer
| |
| - | |-
| |
| - | | 2,2 || Arabinopsis Thaliana cDNA
| |
| - | |-
| |
| - | | 8,9 || nucl.free H<sub>2</sub>O
| |
| - | |-
| |
| - | | 26,1µl total volume
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | ===PCR-program===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PCR-step !! Temperature [°C] !! Time [sec]
| |
| - | |-
| |
| - | | Denaturation || 98 || 30
| |
| - | |-
| |
| - | |"30 cycles start"
| |
| - | |-
| |
| - | | Denaturation || 98 || 10
| |
| - | |-
| |
| - | | Annealing || 62 || 30
| |
| - | |-
| |
| - | | Elongation || 72 || 60
| |
| - | |-
| |
| - | |"30 cycles stop"
| |
| - | |-
| |
| - | | Final Elongation || 72 || 120
| |
| - | |-
| |
| - | | Store || 8 ||
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | ==PCR Purification of ATPCS==
| |
| - | - Kit Purification<br />
| |
| - | - Elutionbuffer: 30µl<br />
| |
| - | <img src="https://static.igem.org/mediawiki/2014/f/f0/Team_berlin_0006.jpg" alt="" /><br/>
| |
| - |
| |
| - | - Final concentration: 69 µg/ml
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/18 Wednesday - PCR of drag outs to separate cells for FieF Knockout </h2>
| |
| - |
| |
| - | Pick up 7 single colonies + 20µl d. H<sub>2</sub>O each (on ice) <br>
| |
| - | --> Labeling: K1, K2, K3, K4, K5, K6, K7 <br>
| |
| - | PCR for each colony with Primer combinations: C1-C3, C2-C4, C1-C5 --> 7x3= 21 Samples<br/>
| |
| - |
| |
| - | ===PCR Assay===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Amount [µl] !! Chemical
| |
| - | |-
| |
| - | | 1 || Sample (Colony)
| |
| - | |-
| |
| - | | 13,5 || d. H<sub>2</sub>O
| |
| - | |-
| |
| - | | 2 || 10x Dream Taq Polymerasebuffer
| |
| - | |-
| |
| - | | 1,2 || 25mM MgCl<sub>2</sub>
| |
| - | |-
| |
| - | | 0,5 || 10µM fwd Primer
| |
| - | |-
| |
| - | | 0,5 || 10µM rev Primer
| |
| - | |-
| |
| - | | 0,5 || 10mM dNTPs
| |
| - | |-
| |
| - | | 0,6 || Taq DNA Polymerasse
| |
| - | |}
| |
| - |
| |
| - | ===PCR Program===
| |
| - | Colony-PCR: Elongation Time : 1,2 Min, Annealing Temp.: 55°C <br>
| |
| - |
| |
| - | ===Gel Electrophoresis===<br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/7/71/Team_berlin_0007.jpg" alt="" /><br/>
| |
| - | ====Result====
| |
| - | RV-strain is not appropriate for Fief-Knockout. Unknown genome leads to unspecific binding of designed primers, too many bonds in gel electrophoresis picture.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/19 Thursday </h2>
| |
| - |
| |
| - | Continue working with colonies: K1, K5, K6, K7 from 18.06.2014<br/>
| |
| - |
| |
| - | PCR-programme:
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Amount [µl] !! Chemical
| |
| - | |-
| |
| - | | 1 || Sample (Colony)
| |
| - | |-
| |
| - | | 13,5 || d. H<sub>2</sub>O
| |
| - | |-
| |
| - | | 2 || 10x Dream Taq Polymerasebuffer
| |
| - | |-
| |
| - | | 1,2 || 25mM MgCl<sub>2</sub>
| |
| - | |-
| |
| - | | 0,5 || 10µM fwd Primer C1
| |
| - | |-
| |
| - | | 0,5 || 10µM rev Primer C4
| |
| - | |-
| |
| - | | 0,5 || 10mM dNTPs
| |
| - | |-
| |
| - | | 0,6 || Taq DNA Polymerasse
| |
| - | |}
| |
| - |
| |
| - | PCR-programme: <br>
| |
| - | Elongation time: 2Min, Annealing Temperature: 56°C<br/>
| |
| - |
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 1st Experiment part 1 of 4 ==
| |
| - |
| |
| - | '''Aim''':
| |
| - | Test the possibility of Magnetization of E.coli cells by the expression of human Ferritin:
| |
| - |
| |
| - |
| |
| - | '''Procedure''':
| |
| - |
| |
| - | To evaluate the effect of Ferritin on the Magnetization different cultures were compared:<br/>
| |
| - |
| |
| - |
| |
| - | For Both RV308 and Nissle
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | !Culture !! 1 !! 2 !! 3 !! 4
| |
| - | |-
| |
| - | |Plasimd || +|| -|| +|| +
| |
| - | |-
| |
| - | |iron || +|| +|| -|| +
| |
| - | |-
| |
| - | |induction || +|| +|| +|| -
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | - Transform the Human Ferritin gen carried on the Plasmid PC514 provided from (iGEM Team of Calgary university, Canada) to E. coli strains
| |
| - | (Nissle and RV308) via electroporation, plate on (Amp) plates for selection.
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/20 Friday Restriction digest of pQE_80L and ATPCS-PCR fragment </h2>
| |
| - |
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''1x'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''5x Mastermix'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''pQE_80L'''
| |
| - | |-
| |
| - | | H2O nucfree||23,1µl||115,5µl||2µl
| |
| - | |-
| |
| - | | 10x green FD buffer||2µl||10µl||2µl
| |
| - | |-
| |
| - | | DNA||2,89µl||14,5µl||14µl
| |
| - | |-
| |
| - | | BamHI||1µl||0µl||1µl
| |
| - | |-
| |
| - | | SacI||1µl||0µl||1µl
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | 5x Mastermix = Mastermix for ATPCS <br />
| |
| - |
| |
| - | Enzymes were added to each single aliqout and not into the Mastermix<br />
| |
| - |
| |
| - |
| |
| - | '''Incubation:''' for 1,5 h at 37 °C<br />
| |
| - |
| |
| - | '''Deactivation:''' 80°C for 5 min<br />
| |
| - |
| |
| - |
| |
| - | ''I used the wrong restriction enzymes as I got confused with the PCR fragment. Digestion with SacI and HindIII is needed not BamHI.
| |
| - | ''Repeat PCR for ATPCS as well as restriction digest!
| |
| - |
| |
| - |
| |
| - |
| |
| - | ==Test expression of Ferritin in RV308 (pSB1C3_Ferrtin) and Nissle (pSB1C3_Ferrtin)==
| |
| - |
| |
| - | 5ml overnight culture used as an inoculum.<br/>
| |
| - |
| |
| - | 4 ml culture were diluted to 40 ml with LB and grown for 1 h at 37°C until an OD600 of 0.7 and 0.8 was reached.<br/>
| |
| - |
| |
| - | Non-induced SDS sample was taken and induced with 1 mM IPTG<br/>
| |
| - |
| |
| - | After expression for 3 h induced SDS sample was taken.
| |
| - | ===SDS PAGE===
| |
| - | Ferritin band ecpected at 21kDa<br />
| |
| - |
| |
| - | '''Key:'''<br />
| |
| - | - 1st band: Nissle (non induced)<br />
| |
| - | - 2nd band: RV308 (non induced)<br />
| |
| - | - 3rd band: Ladder<br />
| |
| - | - 4th band: Nissle (induced)<br />
| |
| - | - 5th band: RV308 (induced)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/25 Wednesday - Amplification of ATPCS (cDNA) </h2>
| |
| - | ===PCR-pipetting scheme===
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Volume [µl] !! Chemicals
| |
| - | |-
| |
| - | | 12,5 || 2x Q5 Mastermix
| |
| - | |-
| |
| - | | 1,25 || 10µM fwd Primer
| |
| - | |-
| |
| - | | 1,25 || 10µM rev Primer
| |
| - | |-
| |
| - | | 1,5 || Arabinopsis Thaliana cDNA
| |
| - | |-
| |
| - | | 8,9 || nucl.free H<sub>2</sub>O
| |
| - | |-
| |
| - | | 25,4µl total volume
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | ===PCR-program===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PCR-step !! Temperature [°C] !! Time [sec]
| |
| - | |-
| |
| - | | Denaturation || 98 || 30
| |
| - | |-
| |
| - | |"30 cycles start"
| |
| - | |-
| |
| - | | Denaturation || 98 || 10
| |
| - | |-
| |
| - | | Annealing || 62 || 30
| |
| - | |-
| |
| - | | Elongation || 72 || 60
| |
| - | |-
| |
| - | |"30 cycles stop"
| |
| - | |-
| |
| - | | Final Elongation || 72 || 120
| |
| - | |-
| |
| - | | Store || 8 ||
| |
| - | |}
| |
| - |
| |
| - | ===PCR Purification===
| |
| - | -Purification Kit <br>
| |
| - |
| |
| - | →DNA concentration measurement showed, that the Absorbance ratio of 260nm:280nm is quite low, which means that targeted DNA got contaminated.
| |
| - |
| |
| - | =='''Preparation of pre-culture'''==
| |
| - | '''Strains:'''<br />
| |
| - | - e.coli nissle<br />
| |
| - | - e.coli nissle + ferritin<br />
| |
| - | - RV308<br />
| |
| - | - RV308 + ferritin<br />
| |
| - |
| |
| - | ===Assay===
| |
| - | - 5ml LB media + 1 picked clone of the pre-day<br />
| |
| - | - Addition of 5µl CN37 to the strains with ferritin<br />
| |
| - |
| |
| - | '''Incubation:''' 37°C; 170rpm; o/n
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/27 Friday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 1st Experiment part 2 of 4 ==
| |
| - |
| |
| - | - Preculture of 1 positive clone over night ( preculture: 7 ml LB+ 7µL Amp + 1 Clone)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/28 Saturday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 1st Experiment part 3 of 4 ==
| |
| - |
| |
| - |
| |
| - | - Transfer the Precultures (OD= 0.4-0.5) to 25, 50 mL flasks (half filled with LB) and cultivate them at 37°C for 3 hours
| |
| - | RV308 (+,+,+) & (-,+,+) And Nissle (+,+,+) were cultivated in 50 mL flasks<br/>
| |
| - |
| |
| - |
| |
| - | Strain: OD<br/>
| |
| - | RV308 0,78<br/>
| |
| - | Nissle 0,86<br/>
| |
| - |
| |
| - | - Induction with (20µl (50 ML flasks)/10µl (25 ML flasks)) IPTG (concentation 1 M).<br/>
| |
| - | - Add FeCl2 and MnCl2 (100µL in 50ml flasks/ 50µl in 25ml flasks) Incubation over night at 37C°<br/>
| |
| - |
| |
| - |
| |
| - | Strain: OD<br/>
| |
| - | RV308 2.56<br/>
| |
| - | Nissle 2.8<br/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/29 Sunday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 1st Experiment part 4 of 4 ==
| |
| - |
| |
| - | -Sampling for testing the Ferritin content via SDS-Page, different ODs were taken in consideration! <br/>
| |
| - |
| |
| - | -Prepare the sample for SDS-gel by centrifuation and discard the supernatant then add (60µl Dis.water + 15 SDS+ MH-EtoH), boil for 10 min at 94°C.<br/>
| |
| - |
| |
| - | - Test the Magnetization using an permanent magnet under light microscope. <br/>
| |
| - |
| |
| - |
| |
| - | '''Evaluation''':
| |
| - |
| |
| - | '''SDS Page''': was not clear!
| |
| - |
| |
| - | '''Magnetization :
| |
| - | '''
| |
| - |
| |
| - | '''RV308'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | !Culture !! 1 !! 2 !! 3 !! 4
| |
| - | |-
| |
| - | |Plasimd || +|| -|| +|| +
| |
| - | |-
| |
| - | |iron || +|| +|| -|| +
| |
| - | |-
| |
| - | |induction || +|| +|| +|| -
| |
| - | |-Magnetization|| yes|| No|| No|| yes (strong)}
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | !Culture !! 1 !! 2 !! 3 !! 4
| |
| - | |-
| |
| - | |Plasimd || +|| -|| +|| +
| |
| - | |-
| |
| - | |iron || +|| +|| -|| +
| |
| - | |-
| |
| - | |induction || +|| +|| +|| -
| |
| - | |-Magnetization|| yes|| No|| No|| yes (strong)}.
| |
| - | '''Nissle'''
| |
| - | In samples with Iron aggregates were observed with strong Mag.effect.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 06/30 Monday - Colony PCR and Ligation ATPCS </h2>
| |
| - |
| |
| - |
| |
| - | ===PCR pipetting scheme===
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Volume [µl] !! Chemicals
| |
| - | |-
| |
| - | | 13,7 || nucl.free H<sub>2</sub>O
| |
| - | |-
| |
| - | | 2 || 10x Dream Taq Buffer
| |
| - | |-
| |
| - | | 1,2 || 25mM MgCl<sub>2</sub>
| |
| - | |-
| |
| - | | 0,5 || 10mM dNTPs
| |
| - | |-
| |
| - | | 0,5 || 10µM fwd Primer
| |
| - | |-
| |
| - | | 0,5 || 10µM rev Primer
| |
| - | |-
| |
| - | | 1 || Sample (Colony)
| |
| - | |-
| |
| - | | 0,6 || Dream Taq DNA Polymerase
| |
| - | |-
| |
| - | |20µl Total Volume
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | ===PCR program===
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PCR-step !! Temperature [°C] !! Time [sec]
| |
| - | |-
| |
| - | | Denaturating || 95 || 180
| |
| - | |-
| |
| - | |''30 cycles start''
| |
| - | |-
| |
| - | | Denaturating || 95 || 30
| |
| - | |-
| |
| - | |Annealing || 55 || 30
| |
| - | |-
| |
| - | |Elongation|| 72 || 120
| |
| - | |-
| |
| - | |'' 30cycles stop''
| |
| - | |-
| |
| - | |Final Elongation || 72 || 600
| |
| - | |-
| |
| - | | Store || 12 ||
| |
| - | |}
| |
| - |
| |
| - | ===Gel Electrophoresis===
| |
| - | 1% Agarose gel with EtBr; 8µl sample; 5µl Ladder<br />
| |
| - | "Key:"<br />
| |
| - | First band= Ladder<br />
| |
| - | Each other band is a different picked clone (10 clones were picked)<br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/0/0a/Team_berlin_0008.jpg" alt="" />
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/01 Tuesday - ATPCS Ligation </h2>
| |
| - |
| |
| - | The ligation of ATPCS was checked with 1% Agarose gel (stained with ethidiumbromid) after colony pcr.
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/02 Wednesday - Knockout of Fief and Fur in RV308 and Nissle </h2>
| |
| - |
| |
| - | To pepare the knockout 5 µl CM solution (pFerritin with canamycin resistance cassette)and 500 µl culture solution were added to 5 ml LB media. For each stain two samples were determined (RV1/2 and N1/2). The samples were incubated at 37°C up to an OD<sub>600</sub> of 0.53-0.55.
| |
| - | After incubation 1 ml of each culture was transfered in sterile eppi and washed for 7 min at 7000 rpm and 4°C. The supernatant was descarded, the pellet was resuspended in 1 ml sterile H<sub>2</sub>O and washed for 7 min at 7000 rpm and 4°C and descarded the supernatant.
| |
| - |
| |
| - | The pellet of N1 was resuspended in 25 µl pRFP (c = 41 ng/µl) and 25 µl sterile H<sub>2</sub>O. The pellets of N2 and RV1/2 were resuspended in 2 µl pRFP and 50 µl sterile H<sub>2</sub>O. 50 µl of each suspension were transfered in electroporation cuvette and electroporated for a few sec (table below). Directly after this, 1 ml fresh LB media (37°C) was added, the suspension was transfered in a sterile eppi and incubated at 37°C at 600 rpm. The cultures were recovered, deleted on CM+Amp plates and incubated at 37°C ove night.
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Überschrift !! [kV] !! [ms]
| |
| - | |-
| |
| - | | N1 || 1,8 || 2,3
| |
| - | |-
| |
| - | | N2 || 1,2 || 5,4
| |
| - | |-
| |
| - | | RV1 || 1,2 || 5,3
| |
| - | |-
| |
| - | | RV2 || 1,5 || 5,3
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | <strong>Transformation of stains RV, Nissle, WM</strong><br/>
| |
| - |
| |
| - | The stains RV308[ferritin], Nissle[ferritin], RV308, Nissle and WM110 were transformed with RFP. RV308[ferritin] and Nissle[ferritin] were deleted on CM+Amp plates and 5 ml culture was added with CM/Amp and incubated at 37°C. The transformed strains RV308, Nissle and WM110 were deleted on Amp plates and 5 ml culture was added with Amp and incubated at 37°C.
| |
| - | <br/>
| |
| - |
| |
| - |
| |
| - | <strong>Results of transformation</strong><br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! stain !! colonies on plate !! cells in culture
| |
| - | |-
| |
| - | | Nissle1 + RFP || 3 || Yes
| |
| - | |-
| |
| - | | Nissle2 + RFP || 19 || Yes
| |
| - | |-
| |
| - | | WM110 1 + RFP || 0 || Yes
| |
| - | |-
| |
| - | | WM110 2 + RFP || n.d. || Yes
| |
| - | |-
| |
| - | | RV308 1 + RFP || 0 || Yes
| |
| - | |-
| |
| - | | RV308 2 + RFP || 87 || Yes
| |
| - | |-
| |
| - | | Nissle[ferritin]1 + RFP || 10<sup>8</sup> || No
| |
| - | |-
| |
| - | | Nissle[ferritin]2 + RFP || 0 || No
| |
| - | |-
| |
| - | | RV308[ferritin] 1 + RFP || 330 || Yes
| |
| - | |-
| |
| - | | RV308[ferritin] 2 + RFP || 300 || Yes
| |
| - | |}
| |
| - |
| |
| - | The cultures of WM110 1 + RFP and WM110 2 + RFP were deleted on Amp plates and incubated at 37°C. Moreover 5 ml precultures from RV308 + RFP + Amp and RV308[ferritin] + RFP + CM+Amp were prepared.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/03 Thursday - PCR amplification of knockout cassette FUR/FieF </h2>
| |
| - |
| |
| - | The pcr preparation was performed at the pattern in the table below.<br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! pcr preparation !! FUR (25µl) !! FieF (50µl)
| |
| - | |-
| |
| - | | Os High Fedility 2x Mastermix || 12,5 µl || 25 µl
| |
| - | |-
| |
| - | | forward primer 10mM || 1,25 µl || 2 µl
| |
| - | |-
| |
| - | | reverse primer 10mM || 1,25 µl || 2,5 µl
| |
| - | |-
| |
| - | | pkD4 plasmid DNA 1 ng/µl || 1 µl || 1 µl
| |
| - | |-
| |
| - | | nuclease-free H<sub>2</sub>O || to 25 µl || to 50 µl
| |
| - | |}
| |
| - |
| |
| - | The amplification was performed with the programm in the table below.<br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! step !! T [°C] !! t [sec]
| |
| - | |-
| |
| - | | initial denaturation || 98 || 30
| |
| - | |-
| |
| - | | 5 cycles || 98/ 63/ 72 || 8/ 25/ 45
| |
| - | |-
| |
| - | | 25 cycles || 98/ 72 || 8/ 70
| |
| - | |-
| |
| - | | final elongation || 72 || 120
| |
| - | |}
| |
| - | --> hold at 8°C
| |
| - |
| |
| - | The amplification results were analysed in agarose gelelectrophorese (1% agarose; 0,5µl Gel Red; 10x diluted ladder; 6x loading dye).
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/04 Friday - Expression von RV308 (RFP) und RV308 (RFP + Ferritin) </h2>
| |
| - |
| |
| - |
| |
| - |
| |
| - | 50 ml LB + 50 µl Amp inoculated with 5 ml overnight culture of RV308 (RFP)<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | 50 ml LB + 50 µl Amp/Cm inoculated with 5 ml overnight culture of RV308 (RFP + Ferritin)<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | After a OD of 0.6-0.7 was reached 50 ml cultures were portioned into 4 sterile 100 ml flasks so that there was a final volume of 10 ml in each flask.<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | The cultures were then cultred the following:<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|'''RV308 (RFP)'''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|'''RV308 (RFP + Ferritin)'''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | | align="center" style="background:#f0f0f0;"|''' '''
| |
| - | |-
| |
| - | | Induced 1 mM IPTG||+||-||+||-||||+||-||+||-
| |
| - | |-
| |
| - | | Fe/Mn each 1 mM*||+||+||-||-||||+||+||-||-
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | *Fe/Mn were solubilized by autoclaving and 1M stock solution<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Altough not planned IPTG and metal ion solutions were added simultaneously due to time constraints. It was planned to add metal ions 2h after induction
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/07 Monday - Preculture prepared of: </h2>
| |
| - |
| |
| - |
| |
| - | 1. ATPCS Klon 1 - 10 (Amp) --> check if anything grew and contact Johann<br/>
| |
| - |
| |
| - | 2. WM110 (RFP), RV308 (RFP), Nissle (RFP), RV308 (Ferritin + RFP) --> make Cryostock for each<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Overgrown WM110 (RFP) plate streaked out again, please take out and put in cool room<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Rune:<br/>
| |
| - |
| |
| - | PCR of PPMT and ATPCS
| |
| - |
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/09 Wednesday - PCR for PPMT with goTaq (Promega) </h2>
| |
| - |
| |
| - |
| |
| - | 25 mikrol reaction:<br/>
| |
| - |
| |
| - | 12,5 mikrol goTaqMastermix<br/>
| |
| - |
| |
| - | 1,25 mikrol fw Primer 10mikrom<br/>
| |
| - |
| |
| - | 1,25 mikrol b Primer 10mikrom<br/>
| |
| - |
| |
| - | 1,0 mikrol DNA Template PPMT <br/>
| |
| - |
| |
| - | 9,0 mikrol nucleasefreies Wasser<br/>
| |
| - |
| |
| - | '''Mit folgenden PCR-Programm Parametern:'''
| |
| - |
| |
| - | Denaturation 98°C 10 min<br/>
| |
| - | Denaturation loop 98°C 1 min<br/>
| |
| - | Annealing Temp loop 56°C 1 min<br/>
| |
| - | Elongation Time loop 72°C 1 min<br/>
| |
| - | Final Elongation 72°C 5 min<br/>
| |
| - | Storage 8°C infinite<br/>
| |
| - |
| |
| - | == Miniprep ==
| |
| - | Miniprep of ATPCS clone number 3 = 94ng/µl and sent for Sequencing
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/17 Thursday - Midiprep </h2>
| |
| - | #pQE_80L = 183 ng/µl<br/>
| |
| - | #pMA-T_PPMT =321ng/µl<br/>
| |
| - | #pKD46 = 218ng/µl<br/>
| |
| - |
| |
| - | == '''<big>Digestion</big>''' ==
| |
| - | Digestion pQE_80L with HindIII and SacI<br>
| |
| - | deactivated at 80°C for 10 mins
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/24 Thursday - miniPrep + restriktion PPMT und iGEM plasmids </h2>
| |
| - |
| |
| - | Plasmide:
| |
| - |
| |
| - | 1. pBADex-mYFP Venus (Amp)<br/>
| |
| - |
| |
| - | 2. pEx-HISII (Amp)<br/>
| |
| - |
| |
| - | 3. pJS418_phagemid(dummy) (Cm)<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Restriction-enzymes: XbaI | PstI (used SpeI instead :X)<br/>
| |
| - |
| |
| - | kit-miniprep<br/>
| |
| - |
| |
| - | 1. 220ng/µl 2. 130ng/µl 3. 430ng/µl<br/>
| |
| - |
| |
| - | restriction:<br/>
| |
| - |
| |
| - | 500 ng DNA + 1µl XbaI + 1µl PstI + 5µl 10x Buffer + ->50µl Wasser (x3) [ppmt x4]<br/>
| |
| - |
| |
| - | 1. 2,3µl 2. 3,8µl 3. 1,2 µl PPMT 1,6µl<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | 2nd try w/ right enzyme:<br/>
| |
| - |
| |
| - | 1,5 µg DNA + 0.5 µl enzymes + 3µl BUffer + -> 30 µl Wasser<br/>
| |
| - |
| |
| - | 1. 6,5µl 2. 11 µl 3. 3,5 µl PPMT 4,5 µl (x3)<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Fragments: 1. 4043/700 2. 4450/55 3. 3800/861 PPMT 2400/251<br/>
| |
| - |
| |
| - | Gel1 L|3| 1 |1|2|2|2|P|3|3 Gel2 L|P|P|P<br/>
| |
| - |
| |
| - |
| |
| - | Neu: Gel 1|2|3|L|P|P|P<br/>
| |
| - |
| |
| - | GelEx QiagenKit -> extrakte in grüner box im freezer<br/>
| |
| - |
| |
| - | Ligation 2 + P<br/>
| |
| - |
| |
| - | 10µl Ansatz: 1µl BUffer + 0.5 µl Ligase + 1.4 µl PPMT + 7.14 µl pEX-HisII [78ng]
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/25 Friday </h2>
| |
| - | 1. Ordered Primer for cloning hu_Ferritin into pQE80L<br/>
| |
| - |
| |
| - | 2.) Ligation of PPMT Fragment from pMAC_PPMT into pEX_HisII<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | pMAC_PPMT and pEX_HisII were digested with HF PstI and HF XbaI (NEB)<br/>
| |
| - |
| |
| - | PPMT (Insert) and cut pEX_HisII (Vector DNA) were purified via an Gel extraction<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | Ligation was performed using NEB T4 DNA Ligase in molar ratios of (Insert:Vector) and 40 ng Vector were used.<br/>
| |
| - |
| |
| - | 0:1 2:1 3:1 5:1<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Ratio'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''02:01'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''03:01'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''05:01'''
| |
| - | |-
| |
| - | | Amount Insert||4,04 ng||6,06 ng||10,1 ng
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Ligase was deactivated and ligation transformed after weekend
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/28 Monday </h2>
| |
| - |
| |
| - | 1. Degradation test of prepped TB-Expression Plasmids by running samples on agarose gel<br/>
| |
| - |
| |
| - | pBADex_MYFP_Venus (no observable degradation)<br/>
| |
| - |
| |
| - | pEX_HisII (no observable degradation)<br/>
| |
| - |
| |
| - | pJS418_Phagemid (Dummy) (no observable degradation)<br/>
| |
| - |
| |
| - | Data:<br/>
| |
| - |
| |
| - | Fabian : 28072014_1<br/>
| |
| - |
| |
| - | 2.Transformation of 0.2 µl pEX_His_PPMT ligation into DH5alpha, DH10b, and MG1655 via electroporation
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/29 Tuesday </h2>
| |
| - |
| |
| - | 1.) Transformation worked and we got colonies for pEX_HisII_PPMT<br/>
| |
| - |
| |
| - | Colony PCR was performed (Sascha)<br/>
| |
| - |
| |
| - | See Standard Protocol<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | 2.) Ligation of ATPCS PCR Fragment into pQE_80L<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | ATPCS PCR fragment and PQE80L Vector were digested with FD HindIII and FD SacI (ThermoScientific)<br/>
| |
| - |
| |
| - | ATPCS was purified via PCR purification and Vector DNA was purified via an Gel extraction<br/>
| |
| - |
| |
| - | Vector DNA was dephosphorylised using Fast-AP and deactivated<br/>
| |
| - |
| |
| - | Ligation was performed using NEB T4 DNA Ligase in molar ratios of (Insert:Vector) and 40 ng Vector were used.<br/>
| |
| - |
| |
| - | 0:1 2:1 3:1 5:1
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''Ratio'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''02:01'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''03:01'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''05:01'''
| |
| - | |-
| |
| - | | Amount Insert||24,96 ng||37,41 ng||62,36 ng
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Ligase was deactivated and ligation transformed next day
| |
| - | <br/><br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/1/11/Team_berlin_0009.jpg" alt="" /><br/>
| |
| - |
| |
| - | #pMAC_PPMT*
| |
| - | #pQE_80L*
| |
| - | #pSB1c3
| |
| - | #pKD46*
| |
| - |
| |
| - | (*)Bands at 20 kbp for Promega prepped Plasmids.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/30 Wednesday</h2>
| |
| - | == Results Colony PCR for pEX-HisII-PPMT clones ==
| |
| - |
| |
| - |
| |
| - | preculture 4 and 5 in incubator for Miniprep and Sequencing<br/>
| |
| - |
| |
| - | == Transformation ==
| |
| - | ===Chemical competent cells XL Blue ===
| |
| - | * 3 µl for each Ligation Assay
| |
| - | * 10 min incubation on ice
| |
| - | *60 s Heat schock
| |
| - | * 2 min incubation on ice
| |
| - | *950µl LB added
| |
| - | *1h --> 37 °C for Recovery
| |
| - | *platted on Amp-plates
| |
| - | === Eporation ===
| |
| - | *0.2 µl Ligationassay in competent DH5α [iGEM]
| |
| - | *1.7 kV
| |
| - | * 1h; 37°C
| |
| - | * platted on Amp plates
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 07/31 Thursday </h2>
| |
| - | Miniprep for sequencing of pEx_His_ppmt in DH10B? 90-100 ng/µl<br/>
| |
| - |
| |
| - | Cryostock & miniprep of pjs418 in DH5a 518ng/µl<br/>
| |
| - |
| |
| - | PCR of 7 clones with pQe80l_ATPCS<br/>
| |
| - |
| |
| - | 16 µl wasser<br/>
| |
| - |
| |
| - | 2 green dreamtaq buffer<br/>
| |
| - |
| |
| - | 0.5 dNTP<br/>
| |
| - |
| |
| - | 0.5 primer (each)<br/>
| |
| - |
| |
| - | colony<br/>
| |
| - |
| |
| - | 0.6 dream taq<br/>
| |
| - |
| |
| - |
| |
| - |
| |
| - | 95° 3min<br/>
| |
| - |
| |
| - | 95° 30s x30<br/>
| |
| - |
| |
| - | 55° 30s x30<br/>
| |
| - |
| |
| - | 72° 2min x30<br/>
| |
| - |
| |
| - | 72° 10min<br/>
| |
| - |
| |
| - |
| |
| - | Colonie PCR did not work--> no positive clones
| |
| - | concentration after MiniPrep<br>
| |
| - | * pJS418 = 518 ng/µl
| |
| - | * Klone 4 = 90 ng/µl
| |
| - | * Klone 5= 105 ng/µl
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/06 Wednesday </h2>
| |
| - |
| |
| - | PCR
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''BamHI_PPMT_GS'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''GS_ATPCS_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|'''BamHI_HuFerritin_HindIII'''
| |
| - | |-
| |
| - | | Q5 2x Mastermix||12,5||12,5||25
| |
| - | |-
| |
| - | | 10 µM Primer fw||1,25||1,25||1,25
| |
| - | |-
| |
| - | | 10 µM Primer rev||1,25||1,25||1,25
| |
| - | |-
| |
| - | | template (1ng)|| 0,30|| 0,5|| 1
| |
| - | |-
| |
| - | | nucfree H2O|| 9,7|| 9,5|| 19
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Program:
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''BamHI_PPMT_GS'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''GS_ATPCS_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''BamHI_HuFerritin_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | 98||30''|| ||98||30''|| ||98||30''
| |
| - | |-
| |
| - | | ||||||||||||||
| |
| - | |-
| |
| - | | 98||10''||||98||10''||||98||10''
| |
| - | |-
| |
| - | | 58||30''||||56,3||30''||||58,6||30''
| |
| - | |-
| |
| - | | 72||12''||||72||60''||||72||45''
| |
| - | |-
| |
| - | | ||||||||||||||
| |
| - | |-
| |
| - | | 72||2'||||72||2'||||72||2'
| |
| - | |-
| |
| - | | 8||end||||8||end||||8||end
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | Analytical Gel
| |
| - | Expacted Bands clearly seen for PPMT and Ferritin. Only little ATPCS band shows Primer Dimer inhibit PCR. Anyhow we will try a Gel extraction and after a Assembly PCR
| |
| - |
| |
| - |
| |
| - | == Generation of Heme-free BFR by Site-directed Mutagenesis, part 1 of 5 ==
| |
| - |
| |
| - | ==== Site-directed mutagenesis was performed by the QuickChange method ====
| |
| - |
| |
| - | Primer design with http://www.bioinformatics.org/primerx/
| |
| - |
| |
| - | methionine (on position 52) was substituted by histidine
| |
| - |
| |
| - | BFR M52H
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/07 Thursday</h2>
| |
| - |
| |
| - | Analysing 2 colonies of pQE80L_ATPCS whether ATPCS was inserted by sequencing revealed no proper results.<br/>
| |
| - |
| |
| - | Tested by colony PCR (see protocols) using 55°C Annealing temp and 2' Elongation time (expected bands should be 2000bps).<br/>
| |
| - |
| |
| - | Gel showed PCR was about 500 bp big -> ATPCS NOT INSERTED.<br/>
| |
| - |
| |
| - | Outcome:BamHI_PPMT_GS super<br>
| |
| - | GS_ATPCS_HindIII bad<br>
| |
| - | BamHI_Ferritin super
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/08 Friday - Gradienten PCR GS_ATPCS_HindIII </h2>
| |
| - | '''PCR'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''3x GS_ATPCS_HindIII'''
| |
| - | |-
| |
| - | | Q5 2x Mastermix||12,5
| |
| - | |-
| |
| - | | 10 µM Primer fw||1,25
| |
| - | |-
| |
| - | | 10 µM Primer rev||1,25
| |
| - | |-
| |
| - | | template (1ng)|| 0,5
| |
| - | |-
| |
| - | | nucfree H2O|| 9,5
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | '''Program:'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''GS_ATPCS_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | 98°C||30''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 98°C||10''||
| |
| - | |-
| |
| - | | 56,3-62°C||30''||34
| |
| - | |-
| |
| - | | 72°C||60''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 72°C||2'||
| |
| - | |-
| |
| - | | 8°C||end||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | - preparative agarose gel 1% and gel ex of GS_ATPCS_HindIII bands at ca. 1490 bp<br/>
| |
| - |
| |
| - | - accidentally also loaded BamHI_PPMT_GS onto Gel and was also extracted (elution with 25 µl elution buffer).<br/>
| |
| - |
| |
| - | - BamHI_Ferritin_HindIII was PCR purified<br/>
| |
| - |
| |
| - | - DNA concentration meassurement for purificated pcr fragments:<br/>
| |
| - |
| |
| - | GS_ATPCS_HindIII = 33 ng/µl<br/>
| |
| - |
| |
| - | BamHI_PPMT_GS = 48 ng/µl<br/>
| |
| - |
| |
| - | BamHI_Ferritin_HindII = 125 ng/µl<br/>
| |
| - |
| |
| - | '''Assembly PCR'''
| |
| - |
| |
| - | An Assembly PCR was used to align BamHI_PPMT_GS with GS_ATPCS_HindIII and amplify the complementary structure in order to form a fusion protein coding sequence.<br/>
| |
| - |
| |
| - | Therefore, both fragments were added a templated equal molar amounts.<br/>
| |
| - |
| |
| - | Calcultation:<br/>
| |
| - |
| |
| - | bpATPCS = 1493<br/>
| |
| - |
| |
| - | bpPPMT = 255<br/>
| |
| - |
| |
| - | mATPCS/bpATPCS = mPPMT/bpPPMT<br/>
| |
| - |
| |
| - | mATPCS = (1ng * 1493) / 255<br/>
| |
| - |
| |
| - | mATPCS= 58 ng<br/>
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|'''BamHI_PPMT_GS_ATPCS_HindIII'''
| |
| - | |-
| |
| - | | Q5 2x Mastermix||25
| |
| - | |-
| |
| - | | 100 µM Primer fw||0.25 (not added till later to second PCR run)
| |
| - | |-
| |
| - | | 100 µM Primer rev||0.25 (not added till later to second PCR run)
| |
| - | |-
| |
| - | | template (1ng)|| 1,750 µl ATPCS + 0.208 µl PPMT
| |
| - | |-
| |
| - | | nucfree H2O|| 18.042
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | '''Program:'''
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''1. PCR BamHI_PPMT_GS_ATPCS_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | 98||30''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 98||10''||
| |
| - | |-
| |
| - | | 68||30''|| 5
| |
| - | |-
| |
| - | | 72||55''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 72||1'||
| |
| - | |-
| |
| - | | 8||end||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | {| {{table}}
| |
| - | | align="center" style="background:#f0f0f0;"|'''2. PCR BamHI_PPMT_GS_ATPCS_HindIII'''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | | align="center" style="background:#f0f0f0;"|''''''
| |
| - | |-
| |
| - | | 98||30''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 98||10''||
| |
| - | |-
| |
| - | | 68||30''|| 30
| |
| - | |-
| |
| - | | 72||55''||
| |
| - | |-
| |
| - | | ||||
| |
| - | |-
| |
| - | | 72||2'||
| |
| - | |-
| |
| - | | 8||end||
| |
| - | |-
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | </gallery>
| |
| - |
| |
| - |
| |
| - | === Transformation into DH10B ===
| |
| - | #BFR M52H
| |
| - | # BFR M52H
| |
| - | *Recover in LB--> 1h at 37°C
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/11 Monday - Plan-prepare fragments for digest </h2>
| |
| - | Constructs aimed for
| |
| - | *In pQE_80L (Amp) BamHI_PPMT_GS_ATPCS_Hind III (PCR Construct available)
| |
| - | Construct Size 1724 of 4731
| |
| - |
| |
| - | *In pQE_80L (Amp) BamHI_FTH_FTL_Hind III (PCR Construct available)
| |
| - | Construct Size 1077 of 4731
| |
| - |
| |
| - | *In pQE_80L (Amp) Sacl_ATPCS_Hind III (PCR Construct not available)
| |
| - | Construct Size 1400 of 4731
| |
| - |
| |
| - | *In pJS418 (CM) XbaI_PPMT_PstI (PCR Construct available)
| |
| - | Construct Size 255 of 3800
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Q5 MasterMix !! 25μl
| |
| - | |-
| |
| - | | ATPCS SacI || 2.5
| |
| - | |-
| |
| - | | ATPCS Hind III|| 2.5
| |
| - | |-
| |
| - | | cDNA At. || 0.5
| |
| - | |-
| |
| - | | H<sub>2</sub>O || 19.5
| |
| - | |-
| |
| - |
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PCR Programm !!
| |
| - | |-
| |
| - | | 98°C|| 30 sec
| |
| - | |-
| |
| - | | 98°C || 10 sec
| |
| - | |-
| |
| - | | 62°C 34cycles || 30 sec
| |
| - | |-
| |
| - | | 72°C || 60 sec
| |
| - | |-
| |
| - | | 72°C|| 2 mins
| |
| - | |-
| |
| - | | 4°C|| Store
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | PCR Fragmente PPMT_GS_ATPCS+ATPCS+Purification (Gelex)<br/>
| |
| - |
| |
| - |
| |
| - | After Gelex:<br/>
| |
| - | PPMT 48ng/μl<br/>
| |
| - |
| |
| - | Ferritin 125ng/μl (Pur from PCR)<br/>
| |
| - |
| |
| - | ATPCS 33ng/μl <br/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/12 Tuesday - Preparing chemically competent cells </h2>
| |
| - | V=100ml, 37°C<br/>
| |
| - | BU36=DH10B
| |
| - |
| |
| - | OD at 600nm=0.461A <br/>
| |
| - | DH10B competent cells<br/>
| |
| - |
| |
| - |
| |
| - | '''''Digestion'''''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Inserts PCR Pur !! JBH1 PPMT_GS_ATPCS 1 !! JBH2 PPMT_GS_ATPCS 2 !! JBH3 Ferritin !! JSH ATPCS
| |
| - | |-
| |
| - | | PCR Fragment || 20|| 20 || 8|| 20
| |
| - | |-
| |
| - | | Buffer || 3 || 3|| 3 || 3
| |
| - | |-
| |
| - | | FD BamHI /FD SacI|| 1|| 1|| 1 || 1
| |
| - | |-
| |
| - | | FD Hind III || 1 || 1 || 1 || 1
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O || 5 || 5 || 17|| 5
| |
| - |
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Vector !! VBH pQE_80L !! VSH pQE_80L SacI !! 2x PJS_418 !! 2x P_Mac_PPMT
| |
| - | |-
| |
| - | | Vector || 5.5 || 5.5|| 1.9|| 3.2
| |
| - | |-
| |
| - | | Enzyme 1|| 1 BamHI || 1 SacI || 1 FDxBa|| 1 FDxBa
| |
| - | |-
| |
| - | | Enzyme 2 || 1 HindIII || 1 HindIII || 1 FD PST || 1 FD PST1
| |
| - | |-
| |
| - | | Buffer || 3|| 3 || 2|| 2
| |
| - | |-
| |
| - | | Fast AP || 1|| 1 || 0 || 0
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O|| 18.5|| 18.5 || ||
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | * 35 ng/L PPMT ATPCS 1
| |
| - | * 15ng/L PPMT ATPCS 2
| |
| - | * 25ng/L ATPCS
| |
| - | * 13ng/L P_Mac_PPMT
| |
| - | * 31ng/L PJS_418
| |
| - | <br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/9/91/Team_berlin_0010.jpg" alt=""/><br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Ligation 5:1 !! JBH1 in VBH !! JBH2 in VBH !! JBH3 in VBH!! JSH in VSH !! PPMT in PJS_418
| |
| - | |-
| |
| - | | 10x T4 DNA ligase Buffer|| 1μl|| 1μl || 1μl || 1μl || 1μl
| |
| - | |-
| |
| - | | Vector || 1.42 || 1.42 || 1.42|| 1.33|| 1.29
| |
| - | |-
| |
| - | | Insert || 3.29 || 7.08 || 1.36 || 7.17 || 1.03
| |
| - | |-
| |
| - | | Nuclease free H<sub>2</sub>O || 3.79 || 0 || 5.72 || 0 || 6.18
| |
| - | |-
| |
| - | | T4 DNA ligase (NEB)|| 0.5 || 0.5 || 0.5 || 0.5 || 0.5
| |
| - |
| |
| - |
| |
| - | |}
| |
| - |
| |
| - | Ampicillin <br>
| |
| - | JBH1 in VBH<br>
| |
| - | JBH2 in VBH <br>
| |
| - | JSH in VSH <br>
| |
| - |
| |
| - | Chloramphenicol <br>
| |
| - | PPMT in PJS_418
| |
| - |
| |
| - | Transformation in DH10b (cc.c)<br>
| |
| - | 10<sup>6</sup> Kolonie for every approach <br>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/13 Wednesday - pQE 80L Colony PCR </h2>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Mastermix !! 1x!! 35x !! 10x
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O|| 14 || 490 || 140
| |
| - | |-
| |
| - | | 10 dreamtaq|| 1 || 70|| 20
| |
| - | |-
| |
| - | | 25µM MgCl<sub>2</sub> || 1.2 || 42|| 12
| |
| - | |-
| |
| - | | 10mM dNTPs || 0.5|| 17.5 || 5
| |
| - | |-
| |
| - | | 10mM Forward Primar || 0.5|| 17.5|| 5
| |
| - | |-
| |
| - | | 10mM Reverse Primar || 0.5 || 17.5 || 5
| |
| - | |-
| |
| - | | Taq|| 0.3 || 10.5 || 3
| |
| - | |
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! PCR Programm !!
| |
| - | |-
| |
| - | | 95°C|| 3 mins
| |
| - | |-
| |
| - | | 95°C || 30 secs
| |
| - | |-
| |
| - | |55°C|| 30 secs
| |
| - | |-
| |
| - | | 72°C|| 60 secs
| |
| - | |-
| |
| - | | 72°C || 10 min
| |
| - | |-
| |
| - | | 8°C|| Store
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Samples : <br>
| |
| - | # PPMT GS ATPCS size 1912
| |
| - | # PPMT GS ATPCS size 1912
| |
| - | # ATPCS in pQE80L size 1770
| |
| - | # Ferritin in pQE80L size 1376
| |
| - | # PPMT in PJS size 420
| |
| - |
| |
| - |
| |
| - | pQE80L=300bp <br>
| |
| - | pJS = 170bp<br>
| |
| - |
| |
| - | Sequence clones A - H + J<br>
| |
| - |
| |
| - |
| |
| - |
| |
| - | == Generation of Heme-free BFR by Site-directed Mutagenesis, part 2 of 5 ==
| |
| - |
| |
| - |
| |
| - | QuickChange Site-Directed Mutagenesis<br>
| |
| - |
| |
| - | 20µL approach:
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Überschrift !! component
| |
| - | |-
| |
| - | | 2 µL || 10x Pfu Buffer (MgSO4)
| |
| - | |-
| |
| - | | 0,6 µL || 10µM forward Primer
| |
| - | |-
| |
| - | | 0,6 µL || 10µM reverse Primer
| |
| - | |-
| |
| - | | x µL || 30ng Template
| |
| - | |-
| |
| - | | 0,4 µL || dNTPs
| |
| - | |-
| |
| - | | 0,6 µL || Pfu Polymerase
| |
| - | |-
| |
| - | | to 20 µL || PCR Wasser
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Template BFR: <br/>
| |
| - |
| |
| - | 0,5 µL A4 (66ng/µL)<br/>
| |
| - |
| |
| - | 0,2 µL D2 (144ng/µL)<br/>
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! labeling !! Template !! Annealing Temp.
| |
| - | |-
| |
| - | | 17 || D2 (144ng/µL) || 71°C
| |
| - | |-
| |
| - | | 15 || D2 (144ng/µL) || 55°C
| |
| - | |-
| |
| - | | 67 || A4 (66ng/µL) || 71°C
| |
| - | |-
| |
| - | | 65 || A4 (66ng/µL) || 55°C
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | PCR Program<br/>
| |
| - |
| |
| - | {|
| |
| - | |-
| |
| - | ! Temp !! time
| |
| - | |-
| |
| - | | 95°C || 30sec
| |
| - | |-
| |
| - | | ________ || ______
| |
| - | |-
| |
| - | | 16 Cyclen ||
| |
| - | |-
| |
| - | | 95°C || 30sec
| |
| - | |-
| |
| - | | 55/71°C || 1min
| |
| - | |-
| |
| - | | 68°C || 5min
| |
| - | |-
| |
| - | | ________ || ______
| |
| - | |-
| |
| - | | 68°C || 10sec
| |
| - | |-
| |
| - | | 4°C || finale
| |
| - | |}
| |
| - |
| |
| - | Σ20µL -8µL fürs Gel= 12µL +1µL DpnI
| |
| - |
| |
| - |
| |
| - | 37°C 90min<br/>
| |
| - |
| |
| - | '''chemical Transformation (Fabian)
| |
| - | '''
| |
| - |
| |
| - | in DH10b<br/>
| |
| - |
| |
| - |
| |
| - | plating out on Amp plates
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/15 Friday - Clone Sequencing </h2>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Clone !! Name !! Construct present !! concentration after isolation
| |
| - | |-
| |
| - | | A|| pQE_80L_PPMT GS ATPCS || yes || 388ng/µL
| |
| - | |-
| |
| - | | B|| pQE_80L_PPMTGSATPCS || yes || 373ng/µL
| |
| - | |-
| |
| - | | C || pQE_80L_PPMTGSATPCS || yes|| 442ng/µL
| |
| - | |-
| |
| - | | D || pQE_80L_PPMTGSATPCS || no|| 389 ng/µL
| |
| - | |-
| |
| - | | E || pQE_80L_huFerritin || yes|| 490ng/µL
| |
| - | |-
| |
| - | | F|| pQE_80L_huFerritin || no|| 402ng/µL
| |
| - | |-
| |
| - | | G || pQE_80L_ATPCS|| yes || 497ng/µL
| |
| - | |-
| |
| - | | H || pQE_80L_ATPCS || yes || 443 ng/µL
| |
| - | |-
| |
| - | | I || pJS418_PPMT|| yes || 596 ng/µL
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/19 Tuesday </h2>
| |
| - |
| |
| - | == Generation of Heme-free BFR by Site-directed Mutagenesis, part 3 of 5 ==
| |
| - |
| |
| - | ÜN culture<br/>
| |
| - |
| |
| - | 5ml LB + 5µL Amp →shakingincubator
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/20 Wednesday </h2>
| |
| - |
| |
| - | == Generation of Heme-free BFR by Site-directed Mutagenesis, part 4 of 5 ==
| |
| - |
| |
| - |
| |
| - | MiniPrep nach Protocol<br/>
| |
| - | Photometer<br/>
| |
| - | 4µL Probe + 76µL dilution (Wasser)
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/21 Thursday </h2>
| |
| - | == Generation of Heme-free BFR by Site-directed Mutagenesis, part 5 of 5 ==
| |
| - |
| |
| - |
| |
| - | Sequencing:
| |
| - | <br />
| |
| - | Primer PB 16
| |
| - | <br />
| |
| - | Crystocks:
| |
| - | <br />
| |
| - | ÜN culture
| |
| - | <br />
| |
| - | 500µL culture + 500µL DMSO<br />
| |
| - | 2xclon 17<br />
| |
| - | 2xclon 67
| |
| - | <br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/6/62/Team_berlin_0011.png" alt=""/><br/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/22 Friday - SDS-PAGE with Coomasie staining </h2>
| |
| - | Samples<br />
| |
| - | # Marker
| |
| - | # Pellet ATPCS/PPMT
| |
| - | #15:00 after induction ATPCS/PPMT
| |
| - | #overnight ATPCS/PPMT
| |
| - | #GS Pellet
| |
| - | #PGSA after induction 15:00
| |
| - | #GS P/A overnight
| |
| - | #human Ferritin Pellet
| |
| - | #human Ferritin after induction 15:00
| |
| - | #human Ferritin overnight
| |
| - |
| |
| - |
| |
| - | '''''Analysis of Expression'''''<br>
| |
| - | Human Ferritin = 42 kDa (Potparam Expasy)<br>
| |
| - | ATPCS = 56.3 kDA (Potparam Expasy)<br>
| |
| - | PPMT = 7.9 kDA (Potparam Expasy)<br>
| |
| - | ATPCSGSPPMT = 64.3 kDa (Potparam Expasy)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 08/29 Friday - Calibration curve for Iron concentration measurement </h2>
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Überschrift !! Überschrift
| |
| - | |-
| |
| - | | 0.017A || 10 µg/ml
| |
| - | |-
| |
| - | | 0.105A || 50 µg/ml
| |
| - | |-
| |
| - | | 1.076A || 100 µg/ml
| |
| - | |-
| |
| - | | 2.095A || 150 µg/ml
| |
| - | |-
| |
| - | | 2.747A || 200 µg/ml
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/03 Wednesday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 1 of 8 ==
| |
| - |
| |
| - | '''Aim''':
| |
| - | Test the possibility of Magnetization of E.coli cells by the expression of human Ferritin:
| |
| - |
| |
| - |
| |
| - | '''Procedure''': ...
| |
| - |
| |
| - |
| |
| - | -Precultures of Nissle and RV308 (wild types)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/04 Thursday </h2>
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 2 of 8 ==
| |
| - |
| |
| - |
| |
| - | -Prepare chemical competent cells ( Nissle and RV308) . see Protocol
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/05 Friday </h2>
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 3 of 8 ==
| |
| - |
| |
| - | -Chemical biotransforamtion (double Trafo, 200ng of each) of, red fluorescence protein carried on plasmid (kan) Human-Ferritin carried on PQE-80L (Amp) [was prepared by iGEM-Berlin using the '''Biobrick''' from Calgary, unlike does not contains polypeptide at the beginning].
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/06 Saturday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 4 of 8 ==
| |
| - |
| |
| - | -Positive colones for both RV308 and Nissle
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/11 Thursday </h2>
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 5 of 8 ==
| |
| - | - Precultures of the transformed colones
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/12 Friday </h2>
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 6 of 8 ==
| |
| - |
| |
| - | - Transfer the precultures into 250ml filled until 100ml with LB and incubate them at 37°C
| |
| - |
| |
| - | Strain OD<br />
| |
| - | RV308 0.71<br />
| |
| - | Nissle 0.6<br />
| |
| - |
| |
| - |
| |
| - | - Induction with 100µl IPTG (1M), incubation over night at '''30°C''' and 200rpm
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/13 Saturday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 7 of 8 ==
| |
| - |
| |
| - | -samples were taken for protein analysis SDS-Page
| |
| - |
| |
| - | Strain OD<br />
| |
| - | RV308 6.2<br />
| |
| - | Nissle 5.2<br />
| |
| - |
| |
| - | - adding 7ml of 1M Mn-citrate (wrongly) and incubated at 4°C over weekend
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/15 Monday </h2>
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, 2nd Experiment part 8 of 8 ==
| |
| - |
| |
| - |
| |
| - | -Test the Magnetization using a permanent magnet under '''Fluorescent microscope.'''
| |
| - |
| |
| - | '''Evaluation'''
| |
| - |
| |
| - | -Cells grew
| |
| - | -no red fluorescence observed
| |
| - | -no Magnetization observed
| |
| - |
| |
| - | Possible Explanation:
| |
| - | - Adding Mn ions instead of Fe could be effected the Magnetization
| |
| - | - The red Fluorescence gen contains a cadaverin chain, this could explain the non-fluorescence.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/16 Tuesday </h2>
| |
| - |
| |
| - | == Preparing Cultures for flouruscence microscopy ==
| |
| - |
| |
| - | E. coli Nissle 1917 (pQE_80L Hu_Ferritin + RFP) <br />
| |
| - | E. coli RV308 (pQE_80L_HuFerritin + RFP)<br />
| |
| - |
| |
| - | Nissle OD600 = 5.08 <br />
| |
| - | RV 308 OD600 = 5.81<br />
| |
| - |
| |
| - | take OD=1<br />
| |
| - |
| |
| - | wash in 1000 µl PBS three times ( centrifuge at 6000g for 2min, discard supertanent and resuspend pellet in PBS)
| |
| - |
| |
| - | finally resuspend pellet in 500 µl
| |
| - |
| |
| - | Put it into a cool box and take it over to the fluorescence microscope facility.
| |
| - |
| |
| - | == Constructing PQE_80L_T5_ATPCS_lac_PPMT ==
| |
| - |
| |
| - | Plasmid for Co-Expression of PPMT and ATPCS on one plasmid.
| |
| - |
| |
| - | Source:
| |
| - | pJS418_PPMT (amplifying insert)
| |
| - | pQE_80L_ATPCS (as target vector)
| |
| - |
| |
| - | === PCR of HindIII_lac_PPMT_HindIII ===
| |
| - |
| |
| - | Construction of the HindIII_lac_PPMT_HindIII in pQE80L_Hind III
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! 3x HindIII_lac_PPMT_HindIII
| |
| - | |-
| |
| - | | Q5 2x Mastermix|| 12,5
| |
| - | |-
| |
| - | | 10 µM Primer fw|| 1,25
| |
| - | |-
| |
| - | | 10 µM Primer rev|| 1,25
| |
| - | |-
| |
| - | | template (1ng)|| 0,5
| |
| - | |-
| |
| - | | nucfree H2O|| 9,5
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | === Program ===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Temperature!! Durattion in seconds!! Cycles
| |
| - | |-
| |
| - | | 98|| 30''|| 1
| |
| - | |-
| |
| - | | 98|| 5''|| loop start
| |
| - | |-
| |
| - | | 62-65|| 15''|| loop 32
| |
| - | |-
| |
| - | | 72|| 10''|| loop end
| |
| - | |-
| |
| - | | 72|| 2'|| 1
| |
| - | |-
| |
| - | | 8|| infinite|| 1
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/17 Wednesday - Digest and Dephosphorylation of vector pQE_80L_ATPCS </h2>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! pQE80L_ATPCS digest!! 1x!! 3x
| |
| - | |-
| |
| - | | pQE_80L_ATPCS|| 2 µl || 7 µl
| |
| - | |-
| |
| - | | FD HindIII|| 1 µl || 3 µl
| |
| - | |-
| |
| - | | 10x FD Buffer|| 2µl || 7 µl
| |
| - | |-
| |
| - | | FastAP (Thermo)|| 1 µl|| 3 µl
| |
| - | |-
| |
| - | | nucfree H2O|| 14 µl || 51 µl
| |
| - |
| |
| - | |}
| |
| - |
| |
| - | Gelextraction resulted in HindIII cut pQE80L_ATPCS 20 µl with 12 ng/µl
| |
| - | <br />
| |
| - |
| |
| - | In parallel the PCR fragment HindIII_lac_PPMT_HindIII was purified and is ready for digest.
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/18 Thursday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 1 of 7 ==
| |
| - |
| |
| - |
| |
| - | ''''''Aim'''
| |
| - | '''
| |
| - |
| |
| - | -Double Trafo of the Human-Ferritin and fluorescence marker.<br />
| |
| - |
| |
| - | '''Procedure:'''…
| |
| - |
| |
| - | Stains: RV308, Nissle and DH0B (control)<br />
| |
| - | -chemical Double-biotransformation of:<br />
| |
| - | -0.5 µl of Human-ferrtin on PQE-80L(490 ng/µl) with (Amp)<br />
| |
| - | -3 µl green fluorescence protein Nerm ASGP-R-GFP (83 ng/µl) with (Kan)<br />
| |
| - |
| |
| - | for control:<br />
| |
| - | - 3 µl green fluorescence protein Nerm ASGP-R-GFP (83 ng/µl) with (Kan) is transformed into DH05<br />
| |
| - | - Double Trafo of ('''pJS418_PPMT and pQE_80L_ATPCS)'''<br />
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/19 Friday </h2>
| |
| - |
| |
| - | '''PCR of BB0 - BB3 parts (Saba)'''
| |
| - |
| |
| - | {| class="wikitable sortable"
| |
| - | |-
| |
| - | ! compound !! V/µl (50µl total)
| |
| - | |-
| |
| - | | Q5 2xMM || 25.0
| |
| - | |-
| |
| - | | 10µM P_fv || 2.5
| |
| - | |-
| |
| - | | 10µM P_rev || 2.5
| |
| - | |-
| |
| - | | DNA (template 1ng)|| 2.0 (of diluted plasmid)
| |
| - | |-
| |
| - | | nuc.-free H2O || 28.0
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | '''Primers'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Reaction !! fw Primer !! rev. Primer !! TmTm/°C !! Template !! bp
| |
| - | |-
| |
| - | | BB0 || BBa_K1438000_fw || BBa_K1438000_rev || 62 || pQE80L_BFR || 507
| |
| - | |-
| |
| - | | BB1 || BBa_K1438000_fw || BBa_K1438000_rev || 62 || pQE80L_BFR M52H || 507
| |
| - | |-
| |
| - | | BB2 || BBa_K1438002_fw || BBa_K1438002_rev || 68 || pQE80L_Hu-Fer || 1000
| |
| - | |}
| |
| - |
| |
| - | '''PCR Programms'''
| |
| - | '''BB0 & BB1'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! T/°C !! t/min !! cycles
| |
| - | |-
| |
| - | | 98 || 0:30 || 30
| |
| - | |-
| |
| - | | 98 || 0:10 || 30
| |
| - | |-
| |
| - | | 62 || 0:30 || 30
| |
| - | |-
| |
| - | | 72 || 0:15 || 30
| |
| - | |-
| |
| - | | 72 || 2:00 || 30
| |
| - | |-
| |
| - | | 8 || infinity || 30
| |
| - | |}
| |
| - |
| |
| - | '''BB2'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! T/°C !! t/min !! cycles
| |
| - | |-
| |
| - | | 98 || 0:30 || 30
| |
| - | |-
| |
| - | | 98 || 0:10 || 30
| |
| - | |-
| |
| - | | 68 || 0:30 || 30
| |
| - | |-
| |
| - | | 72 || 0:40 || 30 (probably too long since ~1000bp and not 1408bp however worked well as seen on gel)
| |
| - | |-
| |
| - | | 72 || 2:00 || 30
| |
| - | |-
| |
| - | | 8 || infinity || 30
| |
| - | |}<br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/9/9c/Team_berlin_0012.jpg" alt=""/><br/>
| |
| - |
| |
| - | Biobricks derived from pQE80L constructs<br>
| |
| - | BB1 BFR 191µg/ml<br>
| |
| - | BB2 BFRM 173µg/ml <br>
| |
| - | BB2 HuFerritin 166 µg/ml
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 2of 7 ==
| |
| - | -No positive colones were detected for double-trafo expect these for control (transformation was successful in DH05).
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/20 Saturday - Digest of PSB1C3-Ferritin </h2>
| |
| - |
| |
| - | ===== Plasmid: PSB1C3 - ferritin with Peptid 120ng/µl =====
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! component !! µL
| |
| - | |-
| |
| - | | NEB Xba1 || 2 µL
| |
| - | |-
| |
| - | | NEB Pst1 || 2 µL
| |
| - | |-
| |
| - | | NEB Buffer 3.1 || 20 µL
| |
| - | |-
| |
| - | | nucleasefree H<sub>2</sub>O || 151 µL
| |
| - | |-
| |
| - | | PSB1C3 Feriitin || 25 µL
| |
| - | |-
| |
| - | | total volume || 200µL
| |
| - | |}
| |
| - |
| |
| - | 37°C for 1,5h
| |
| - | (inactivate for 20min at 80°C)
| |
| - |
| |
| - |
| |
| - | ==== Gel electrophoresis ====
| |
| - | 1% Agarose-Gel <br/>
| |
| - | + 3 µL EthBr<br/>
| |
| - |
| |
| - | 200µL Probe + 40 µL loading dye (6x)<br/>
| |
| - |
| |
| - | 5 µL GenRuler-Mix<br/>
| |
| - |
| |
| - | no Gel-extraction, because nothing was seen on the gel, except the marker (GenRuler) <br/>
| |
| - |
| |
| - | (add Figure)<br/>
| |
| - | Figure not in Dropbox<br/>
| |
| - |
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 3 of 7 ==
| |
| - | -Transformation of RF '''(red fluorescence protein)''' on with (Kan) into DH05
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/21 Sunday </h2>
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 4 of 7 ==
| |
| - |
| |
| - | -Transformation was successful
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/22 Monday - Precultures </h2>
| |
| - |
| |
| - | Precultures of
| |
| - |
| |
| - | PSB1K3_RFP in DH10b (Kan)<br />
| |
| - | pQE80L_ATPCS & pJS418_PPMT in Dh10b (Amp & Cm)<br />
| |
| - | Clones with pQE80L_ATPCS'n'PPMT in DH10b (Amp)<br />
| |
| - | PSB1C3_Ferritin in DH10b (Cm)
| |
| - |
| |
| - | == Checking Insertion of gene by colony PCR ==
| |
| - | Performing PCR for clones of ATPCS-PPMT construct<br>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! !! 1x in µl !! 12x in µl !! 10x in µl
| |
| - | |-
| |
| - | | nuclease free H<sub>2</sub>O|| 14|| 168 || 140
| |
| - | |-
| |
| - | | 10x DreamTaq buffer|| 2 || 24 || 20
| |
| - | |-
| |
| - | | 25mM MgCl<sub>2</sub>|| 1.2|| 14.4|| 12
| |
| - | |-
| |
| - | | 10mM dNTPs || 0.5|| 6 || 5
| |
| - | |-
| |
| - | | 10mM FW Primar<br>HindIII-lac-prom-fw || 0.5 || 6 || 5
| |
| - | |-
| |
| - | | 10mM RW Primar<br> PB17 (T7 term_rev) || 0.5 || 6 || 5
| |
| - | |-
| |
| - | | Taq Polymerase || 0.3 || 3.6 || 3
| |
| - | |}
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Programm colony PCR !! Temp !! Time
| |
| - | |-
| |
| - | | Denaturing|| 95°C|| 7 min
| |
| - | |-
| |
| - | | Denaturing|| 95°C|| 30
| |
| - | |-
| |
| - | | Annealing || 69°C || 30
| |
| - | |-
| |
| - | | Elongation || 72°C || 1 min
| |
| - | |-
| |
| - | | Final Elongation || 72°C || 10 min
| |
| - | |-
| |
| - | | Store|| 8°C ||
| |
| - |
| |
| - | |}
| |
| - |
| |
| - | Dissolved colonies 4 and 6 which showed band at 400 bp were grown each in a cultivation tube with LB+Amp100 at 37°C
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/23 Tuesday - Mutagenesis of ATPCS </h2>
| |
| - |
| |
| - | The elimination of the XbaI restriction site of all our ATPCS containing constructs was achieved by quick change mutagenesis.<br/>
| |
| - |
| |
| - | ... Details Saba....
| |
| - |
| |
| - | After digest with DpnI the reaction was transformed into competent DH10b E. coli cells via heat shock transformation.<br />
| |
| - | After streaking out all aliquots on LB-Amp Agar plates, they were incubated at 37 °C over night.<br/>
| |
| - |
| |
| - | == Digest of miniprepped PSB1C3_Ferritin (Calgary) for extraction of vector for BioBrick preparation ==
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Components !! Volume in µl
| |
| - | |-
| |
| - | | NEB XbaI|| 2
| |
| - | |-
| |
| - | | NEB PstI || 1,5
| |
| - | |-
| |
| - | | NEB Buffer 3.1 || 5
| |
| - | |-
| |
| - | | nucfree H2O || 26,5
| |
| - | |-
| |
| - | | plasmid PSB1C3_ferritin (Calgary) || 15
| |
| - | |-
| |
| - | | || 50 µl reaction
| |
| - | |}
| |
| - | <br />
| |
| - | The reaction was incubated at 37 °C for 1,5 h.<br />
| |
| - |
| |
| - |
| |
| - | The digested vector was purified using a 1% agarose gel and the Roth GelNebulizer purifiction kit. The vector band was expected at 2000 bp and the insert band at about 1000 bp.<br/>
| |
| - |
| |
| - | '''both colonies from Saba were grown --> Mini-prep (Johann) to harvest plasmid DNA'''
| |
| - |
| |
| - | Mutagenesis of ATPCS
| |
| - | in
| |
| - | pQE80L_ATPCS-GS-PPMT<br/>
| |
| - | pQE80L_ATPCS_PPMT<br/>
| |
| - | pQE80L_ATPCS<br/>
| |
| - | 3x PCR-Reactions<br/>
| |
| - | Q5 MM 12.0µl<br/>
| |
| - | PB_for SN_ATPCS_xbaI_Mut_for 1.25µl<br/>
| |
| - | PB_rev SN_ATPCS_xbaI_Mut_rev 1.25µl<br/>
| |
| - | nuc.-free H2O 10.5µl<br/>
| |
| - | template (1ng) ~ 1.0µl (dilations of original plasmids)<br/>
| |
| - | Total 26µl<br/>
| |
| - |
| |
| - | '''PCR Programm'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! T/°C !! t/min
| |
| - | |-
| |
| - | | 98 || 0:30
| |
| - | |-
| |
| - | | 98 || 0:10
| |
| - | |-
| |
| - | | 69 || 0:30
| |
| - | |-
| |
| - | | 72 || :
| |
| - | |-
| |
| - | | 72 || 2:00
| |
| - | |}
| |
| - |
| |
| - | Trafo of 5µl PCR-Product in DH10b cells --> incubation at 37 °C
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 5 of 7 ==
| |
| - |
| |
| - | - Preculture of DH05 with RF plasimd
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/24 Wednesday </h2>
| |
| - |
| |
| - | == Berlin Vector - pQE-80L-JBFS-huFerritin Assembly PCR ==
| |
| - |
| |
| - | ==== Running Gel Electrophoresis ====
| |
| - |
| |
| - | * 1% Agarose, Ethidium bromide
| |
| - |
| |
| - | * 50µL Assembly PCR product + 10µL 6*DNA dye
| |
| - | * 10µL GeneRuler DNA Ladder Mix (ThermoScientific)
| |
| - | * Gel Electrophoresis conditions 100V, 30min
| |
| - | <br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/c/c9/Team_berlin_0013.jpg" alt=""/><br/>
| |
| - |
| |
| - | ==== Gel Extraction ====
| |
| - |
| |
| - | Gel Extraction using GeneJET Gel Extraction Kit (ThermoScientific)
| |
| - |
| |
| - | * Determination of Concentration by Ultraviolet Spectrophotometry
| |
| - |
| |
| - | ?
| |
| - |
| |
| - | ==== Double Digest ====
| |
| - |
| |
| - | * Digestion of 1μg of purified Insert DNA using FastDigest BamHI / HindIII
| |
| - | * 1h at 37°C
| |
| - | ?
| |
| - |
| |
| - | * Heat inactivation of FastDigest BamHI / HindIII, 15min at 80°C
| |
| - |
| |
| - | ==== PCR purification ====
| |
| - |
| |
| - | * PCR purification of BamHI / HindIII digested insert using GeneJET PCR Purification Kit (ThermoScientific)
| |
| - | * Determination of Concentration by Ultraviolet Spectrophotometry
| |
| - |
| |
| - | ?
| |
| - |
| |
| - | ==== Ligation ====
| |
| - |
| |
| - | * Using the Molar Ligation Ratio 1:5 (1 times vector and 5 times insert)
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Position !! Substance !! Volume
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel
| |
| - | |}
| |
| - |
| |
| - | * 20min at RT
| |
| - |
| |
| - | ==== Chemical Transformation ====
| |
| - |
| |
| - | * 100µL Aliquots of Chemically Competent Cells - DH10B ''E.coli'' + 10µL Ligation approach chilled on ice for 30 min
| |
| - | * Heat shock for 1min at 42°C
| |
| - | * Add 900µL preheated (37°C) LB
| |
| - | * Incubate the cells for 1h at 37°C with shaking approx. 200rpm
| |
| - | * Plate 200µL and spin down the rest and plate on LB Ampicillin, incubate overnight at 37°C
| |
| - |
| |
| - | ====Saba====
| |
| - | o/n culture of clones from successful pQE80L_ATPCS-mut_GS_PPMT and pQE80L_ATPCS-mut_PPMT transformation
| |
| - | --> 37°C, 200pm, o/n 5-6 nl LB+AB (Amp)
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 6 of 7 ==
| |
| - |
| |
| - |
| |
| - | - Miniprep of the Preculture, DNA concentration 71 ng/µl
| |
| - |
| |
| - | '''Chemical Biotransformation of:
| |
| - | -'''3 µl of RFP 71 ng/µl'''
| |
| - | -2 µl '''Ferritin jbfs_m (different ferrtin)(120 ng/µl) with (Amp)''''''
| |
| - |
| |
| - |
| |
| - | in RV308 and Nissle (2 plates each)
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/25 Thursday - PCR of Biobrick Parts </h2>
| |
| - | ==Isolation of plasmid-DNA with Mini-Prep Kit of pQE80L_ATPCSMut_GS_PPMT and pQE80L_ATPCS_PPMT==
| |
| - |
| |
| - | '''PCR of Biobrick-Parts 03,04,06 & 10-15'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! BB !! PB_fw !! PB_rew !! Tm/°C !! template !! bp !! part
| |
| - | |-
| |
| - | | 03 || BBa-K1438003_fw || BBa-K1438003_rew || 67 || pQE80L_PPMT_GS_ATPCS || 1715 || PPMT GS ATPCS
| |
| - | |-
| |
| - | | 04 || BBa-K1438004_fw || BBa-K1438003_rew || 65 || pQE80L_PPMT_ATPCS || 1408 || ATPCS
| |
| - | |-
| |
| - | | 10 || XbaI_T7_prom || for Lambda_Term_suffix_rew || 66 -->67 || pQE80L_BFR || 690 (+377bp-->cp) || BFR_cp
| |
| - | |-
| |
| - | | 11 || " || " || 66 -->67 || pQE80L_BFRM52H || 690 (+377bp) || BFRM52H_cp
| |
| - | |-
| |
| - | | 12 || " || " || 66 -->65 || pQE80L_FTH_FLH(HuFer) || 1337(+377bp) || FTH_FLH_cp
| |
| - | |-
| |
| - | | 13 || " || " || 66 -->67 || pQE80L_PPMT_GS_ATPCS || 1968(+377bp) || PPMT_GSATPCS_cp
| |
| - | |-
| |
| - | | 14 || " || " || 66 -->67 || pQE80L_PPMT_ATPCS || 2091(+377bp) || ATPCS_cp
| |
| - | |-
| |
| - | | 06 || " || " || 66 || pQE80L_fbfs_mil_Ferritin || 1403(+377bp) || fbfs_mil_Ferritin_cp
| |
| - | |}
| |
| - |
| |
| - | '''PCR-Ansätze V/µl'''
| |
| - | Q5 2xMM 25.0
| |
| - | 10µM PB_fw 2.5
| |
| - | 10µM PB_rew 2.5
| |
| - | template 2.0 (~1ng)
| |
| - | nuc.-free H2O 18.0
| |
| - | '''PCR-Programm'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! T/°C !! t/min !! cycles
| |
| - | |-
| |
| - | | 98 || 30" || 30
| |
| - | |-
| |
| - | | 98 || 10" || "
| |
| - | |-
| |
| - | | 66/67 || 30" || "
| |
| - | |-
| |
| - | | 72 || 40"/12"/1' || "
| |
| - | |-
| |
| - | | 72 || 2' || "
| |
| - | |-
| |
| - | | 8 || infinity || "
| |
| - | |}
| |
| - | <gallery>
| |
| - | Datei:140925 SN iGEM biobricks4-12.JPEG|PCR Results
| |
| - | </gallery>
| |
| - | PCR purification of 1,2, 3 (Jbfs_mil_Ferritin_cp),BF-cp and BFM-cp biobrick parts
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! !! Concentration µg/ml
| |
| - | |-
| |
| - | | fbfs_mit_Ferritin1 || 42
| |
| - | |-
| |
| - | | fbfs_mit_Ferritin2 || 35
| |
| - | |-
| |
| - | | fbfs_mit_Ferritin3 || 42
| |
| - | |-
| |
| - | | ATPCS and PPMT || -12 too low
| |
| - | |-
| |
| - | | Bacterioferritin CP|| 56
| |
| - | |-
| |
| - | | BFM52H CP || 57
| |
| - | |}
| |
| - |
| |
| - | == Magnetization of E. coli by expression of human Ferritin, Bio-transformations, 3rd Experiment part 7 of 7 ==
| |
| - | - Double-trafo was successful in all RV308 plates but not in Nissle
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 09/30 Tuesday </h2>
| |
| - | ==Ligation of Biobrick parts with PSBIC3 backbone (digested on 23.09.2014 by Johann==
| |
| - |
| |
| - |
| |
| - | Transformation into DH10b c.c.cells by chemical transformation<br/>
| |
| - |
| |
| - | 5µl of each ligation was transformed to 100µl aligusted cells each<br/>
| |
| - |
| |
| - | --> after recovery at 37°C, 30min cells were plated on Cm/LB-Agar plates after centrifuging-->pelleting and resuspending in 100µl LB to plate all cells.
| |
| - |
| |
| - | --> incubation at 37°C, o/n
| |
| - |
| |
| - | == Mini Prep von pSB 1U 3-RFP ==
| |
| - | concentration 112 ng/µl<br>
| |
| - |
| |
| - | Preculture <br>
| |
| - | pQE-80L-jbfs-Ferritin 5ml LB+5µl Amp at 37°C overnight
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/01 Wednesday - Plasmid Digestion & PCR Probe </h2>
| |
| - |
| |
| - | ==== Plasmid Digestion ====
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Plasmid Digestion !!
| |
| - | |-
| |
| - | | NEB Buffer 3.1 || 5µl
| |
| - | |-
| |
| - | | NEB XbaI || 0.5µl
| |
| - | |-
| |
| - | | NEB PstI || 0.5µl
| |
| - | |-
| |
| - | | P_MA_T_PPMT 312ng/µl || 3.2µl
| |
| - | |-
| |
| - | | null. free water || 40.5µl
| |
| - | |}
| |
| - |
| |
| - | ==== PCR Probe ====
| |
| - |
| |
| - |
| |
| - | ===Ligation plates evaluated:===
| |
| - |
| |
| - | All plates had clones except for BFR_cp from each plate transformation one clone was cultivated with LB/Cm (~5ml) o/n at 37°C
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/02 Thursday - Cultures were Mini-preped </h2>
| |
| - |
| |
| - | --> elution with 40µl EB
| |
| - |
| |
| - | --> DNA contration measurement
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/06 Monday- Colony PCR </h2>
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Biobrick !! Sequenzierung !! Sequenz (?) !! (???)
| |
| - | |-
| |
| - | | BB0 || BFR || 100Y BFR M52H || -A ws ATGf (??)
| |
| - | |-
| |
| - | | BB1 || BFR M52H || 100% BFR || -A wr
| |
| - | |-
| |
| - | | BB2 || HFTN_LFFN (?) || 96Y HuFerritin (Rw Sep nötig) ?? ||
| |
| - | |-
| |
| - | | AnP || ATPCS || f 70% ok, aber Fragment f || PCR nochmal ... Gelex (?)
| |
| - | |-
| |
| - | | BFM1cp || BFR1cp || ? ||
| |
| - | |-
| |
| - | | BFM2cp || BFR2cp || ? ||
| |
| - | |-
| |
| - | | HuFecp || HuFerritin cp || ? ||
| |
| - | |}
| |
| - |
| |
| - | ===Colony-PCR of other clones===
| |
| - |
| |
| - | '''PCR-Programm'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! T/°C !! t/min !! cycles
| |
| - | |-
| |
| - | | 95 || 7' || 30
| |
| - | |-
| |
| - | | 95 || 30" || 30
| |
| - | |-
| |
| - | | 52 || 30" || 30
| |
| - | |-
| |
| - | | 72 || 1' || 30
| |
| - | |-
| |
| - | | 72 || 10" || 30
| |
| - | |-
| |
| - | | 8 || infinity || 30
| |
| - | |}
| |
| - |
| |
| - | --> gel (1% Agarose)
| |
| - |
| |
| - | Clones 3, 5, 6, 7 and 13 were grown o/n in LB/Cm for Mini-prep on the following day. therefore 5µl of the disolved clones were used for each inoculation
| |
| - |
| |
| - | --> 37°C, 200rpm
| |
| - |
| |
| - |
| |
| - |
| |
| - | ==2x M9 Media Production==
| |
| - | (Christina)<br />
| |
| - |
| |
| - | 2X M9 mineral medium<br/>
| |
| - |
| |
| - | For 500 ml M9 minearal medium add to XXX ml sterile water:<br/>
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | | 100ml || M9 salt solution (10X) || Na<sub>2</sub>HPO<sub>4</sub> <br />
| |
| - | KH<sub>2</sub>PO<sub>4</sub><br />
| |
| - | NaCl<br />
| |
| - | NH<sub>4</sub>Cl || 67.4mM<br />
| |
| - | 44.0mM<br />
| |
| - | 17.10mM<br />
| |
| - | 18.70mM
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |-
| |
| - | | Beispiel || Beispiel || Beispiel || Beispiel
| |
| - | |}
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/07 Tuesday </h2>
| |
| - | ===Mini-Prep of cultures (Ben) and preparation of sequencing samples (Saba):===
| |
| - |
| |
| - | 9µl nuc.-free H2O + 3µl DNA + 3µl PB794<br>
| |
| - | <br/>
| |
| - | <img src="https://static.igem.org/mediawiki/2014/c/c3/Team_berlin_0014.jpg" alt=""/><br/>
| |
| - |
| |
| - | == Preculture of Knockouts ==
| |
| - | * λΔ FieF +PCP 20 : 5ml LB+tet/amp (1:1000)
| |
| - | * λΔ FUR : 5ml LB+tet/kan (1:1000)
| |
| - | * MG1655 ΔFieF: 5ml LB + amp/kan (1:1000)<br>
| |
| - | incubation at 30°C
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/08 Wednesday - PCR of PSB1C3 Backbone </h2>
| |
| - |
| |
| - | Q5 Mastermix 2x
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Component !! Volume in µl
| |
| - | |-
| |
| - | | Q5 Mastermix 2x || 25
| |
| - | |-
| |
| - | | Primer fw (PSB1C3 Suffix fw)|| 2,5
| |
| - | |-
| |
| - | | Primer rev (PS1C3 Prefix rev|| 2,5
| |
| - | |-
| |
| - | | template (PSB1C3_BBa_K1189018)|| 2
| |
| - | |-
| |
| - | | nuc free H2O|| 18
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Temperature in °C!! Time!! Nr. of cyles
| |
| - | |-
| |
| - | | 98|| 30''||
| |
| - | |-
| |
| - | | 98|| 5''|| loop start
| |
| - | |-
| |
| - | | 70|| 15''|| 30
| |
| - | |-
| |
| - | | 72|| 1'|| loop end
| |
| - | |-
| |
| - | | 72|| 2'||
| |
| - | |-
| |
| - | | 8|| infinit||
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | == Extraction of PCR Product (PSB1C3 linearised) ==
| |
| - |
| |
| - | After extracting the expected band at 2000 bp it was purified using the EMDMillipore Montage Gel Extraction Device.<br>
| |
| - | Resulting in about 120 µl DNA extract in TAE buffer with an concentration of 9ng/µl<br>
| |
| - |
| |
| - |
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Digestion of PCR Product !!
| |
| - | |-
| |
| - | | NEB Buffer 3.1 || 15µl
| |
| - | |-
| |
| - | | NEB PstI|| 1.5µl
| |
| - | |-
| |
| - | | NEB XbaI || 1.5µl
| |
| - | |-
| |
| - | | nuc. free water || 83µl
| |
| - | |-
| |
| - | | PCR Product || 100µl
| |
| - | |}
| |
| - |
| |
| - | * 1.5 hours at 37°C
| |
| - | * PCR Purification
| |
| - | * DNA concentraion measurement = 35ng/µl
| |
| - |
| |
| - | == Ligation ==
| |
| - | Calculation
| |
| - | *BFM52H = 3.22µl
| |
| - | *BF_cp= 6.7µl
| |
| - | *jbfs_m = 8.06 µl
| |
| - | *BB_AnP = 10.64 µl
| |
| - | * HuFer = 9.45 µl
| |
| - | == Iron Concentration measurement ==
| |
| - | 278 mg FeSO<sub>4</sub>.7H<sub>2</sub>O dissolved in 10 ml d.H<sub>2</sub>O <br>
| |
| - | λ FieF PCP<sub>2</sub> Amp 1:100 ---> 3.0 OD<br>
| |
| - | iGEM BV125 tet 1:100 --> 3.0 OD <br>
| |
| - | λ FieF PCP<sub>1</sub> Amp 1:20 -->3.26 OD
| |
| - | iGEM BV125 tet 1:20---> 2.44<br>
| |
| - | too high OD!
| |
| - |
| |
| - | Both samples diluted 1:2
| |
| - | Samples
| |
| - | # FieF + 5µl 0.1 M Fe-stock solution
| |
| - | # FieF + 15 µl 0.1 M Fe-Stock solution
| |
| - | # BV + 5 µl 0.1M Fe-stock solution
| |
| - | # BV + 15 µl 0.1 M Fe-stock solution
| |
| - |
| |
| - | Overnight incubation at 30°C on a shaker
| |
| - | </div>
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/09 Thursday </h2>
| |
| - | Mini Prep (using: AccuPrep Plasmid Mini Extraction Kit Ver. 2.0, Company: BIONEER), using 100µl Elution Buffer <br>
| |
| - | and dsDNA Measurement: 4 µl Sample + 76µl MilliQ, blanked with MilliQ
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Samplenumber !! Samplename !! dsDNA conc [ng/µl] !! Sample [µl] used for Sequencing !! nucl.free Water [µl] used for Sequencing
| |
| - | |-
| |
| - | | #1 || cp HuF || 111 || 9 || 3
| |
| - | |-
| |
| - | | #2 || AGSP || 107 || 9,4 || 2,6
| |
| - | |-
| |
| - | | #3 || AGSP || 59 || 12 || 0
| |
| - | |-
| |
| - | | #4 || AGSP || 64 || 12 || 0
| |
| - | |-
| |
| - | | #5 || jbfs_Fer || 54 || 12 || 0
| |
| - | |-
| |
| - | | #6 || A'n'P || 67 || 12 || 0
| |
| - | |}
| |
| - |
| |
| - | For Sequencing:
| |
| - | *total volume: 15µl
| |
| - | *Primer used: PB795 3µl each Sequencing approach
| |
| - | *Sample [µl] see table
| |
| - | *nucl.free Water [µl] see table
| |
| - |
| |
| - | evaluation of Trafos ---> no growth --->Repeat Trafo in other cells
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/10 Friday - STRATEGY 1 - Knockout of FieF and FUR Genes </h2>
| |
| - | === Detection PCR ===
| |
| - | kan-deletion-cassette for FieF and FUR Genes was transformed into 2 different strains (MG1655 and λ MAGE). Additionally pCP20 was already transformed into one of the λ MAGE Clones (has to be re-validated).<br />
| |
| - | The positive clones have to be validated.<br />
| |
| - |
| |
| - | Detection-PCR of one clone of Plates 1-3:<br />
| |
| - |
| |
| - | Plate 1: MG1655 ΔFieF<br />
| |
| - | Plate 2: λ ΔFieF + pCP20<br />
| |
| - | Plate 3: λ ΔFUR<br />
| |
| - |
| |
| - | === Primer-Combinations ===
| |
| - | '''FieF'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | !- !! Combination !! WT length !! Clone length !! Annealing Temperature range
| |
| - | |-
| |
| - | | A ||C1+C2 || 2060bp || 2654bp || 58,4-60,1°C
| |
| - | |-
| |
| - | | B ||C1+C3 || 0 || 1046bp || 59,5-60,1°C
| |
| - | |-
| |
| - | | C ||C4+C2 || 0 || 1181bp || 57,5-58,4°C
| |
| - | |-
| |
| - | | D ||C1+C5 || 1134bp || 0 || 60,1-60,5°C
| |
| - | |}
| |
| - | --> Annealing Temperature: 57°C<br />
| |
| - |
| |
| - | '''FUR'''
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !!Combination !! WT length !! Clone length !! Annealing Temperature range
| |
| - | |-
| |
| - | | A ||C1+C2 || 1130bp || 2219bp || 59,5°C
| |
| - | |-
| |
| - | | B ||C1+C3 || 0 || 823bp || 59,5°C
| |
| - | |-
| |
| - | | C ||C4+C2 || 0 || 969bp || 57,5-59,5°C
| |
| - | |-
| |
| - | | D ||C1+C5 || 500bp || 0 || 59,5°C
| |
| - | |}
| |
| - | --> Annealing Temperature: 57°C
| |
| - | === PCR-Assay ===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! - !! 1x !! Mastermix 15x
| |
| - | |-
| |
| - | | nuc.free H<sub>2</sub>O || 14,4µl || 216µl
| |
| - | |-
| |
| - | | 10x Dream Taq Buffer || 2µl || 30µl
| |
| - | |-
| |
| - | | 50mM MgCl<sub>2</sub> || 0,6µl || 9µl
| |
| - | |-
| |
| - | | 10mM dNTPs || 0,5µl || 7,5µl
| |
| - | |-
| |
| - | |
| |
| - | |-
| |
| - | | 10mM Primer Combination || 1µl
| |
| - | |-
| |
| - | | 10µl nuc.free H<sub>2</sub>O+ picked colony|| 1µl + 18,4ml Mastermix
| |
| - | |-
| |
| - | | Taq Polymerase || 0,5µl
| |
| - | |}
| |
| - |
| |
| - | === PCR-Program ===
| |
| - | {| class="wikitable"
| |
| - | |-
| |
| - | ! Temperature !! Time
| |
| - | |-
| |
| - | | 95°C || 7'
| |
| - | |-
| |
| - | |30 cycles
| |
| - | |-
| |
| - | | 95°C || 30"
| |
| - | |-
| |
| - | | 57°C || 30"
| |
| - | |-
| |
| - | | 72°C || 2'40"
| |
| - | |-
| |
| - | |
| |
| - | |-
| |
| - | | 72°C || 5'
| |
| - | |-
| |
| - | | 12°C || hold
| |
| - | |}
| |
| - | === Gel-Electrophoresis ===
| |
| - | 1% Agarose-Gel + 1µl GelRed; 90V; 30min<br />
| |
| - | <img src="https://static.igem.org/mediawiki/2014/9/92/Team_berlin_0015.jpg" alt=""/>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="sub-content-project">
| |
| - | <h2 class="sub-content-project-headline"> 10/14 Tuesday - Iron-Assay </h2>
| |
| - | 1. Day: inoculation of 5ml LB Media (+ Antibiotics)<br />
| |
| - |
| |
| - | * 37°C or 30°C/ON<br />
| |
| - | <br />
| |
| - | 2.Day:inoculation of 10ml LB (1:10)<br />
| |
| - | * 30°C or 37°C<br />
| |
| - | * grow until OD<sub>600</sub> 0,6<br />
| |
| - | * take 1ml<br />
| |
| - | * centrifuge 2min at max. speed<br />
| |
| - | * discard the supernatant <br />
| |
| - | * wash with 1X M9 Media<br />
| |
| - | <br />
| |
| - | * 1XM9:<br />
| |
| - | :500µL 2X M9+<br />
| |
| - | :500µL sterile water<br />
| |
| - | * wash 2x and recover 1mL 1XM9 Media<br />
| |
| - | * diluted 1:1000 with 1X M9<br />
| |
| - | * plate 20µL per well
| |
| - |
| |
| - |
| |
| - |
| |
| - | 24well plate without Antibiotics
| |
| - | <br />
| |
| - | *A:MG1655 wt
| |
| - | *B:Nissel 1917 wt
| |
| - | *C:RV308 wt
| |
| - | *D:DH10b
| |
| - | <br />
| |
| - | concentration of Fe-citrat
| |
| - | <br />
| |
| - | 0 0,5 1 2 5 10mM
| |
| - | </div>
| |
| - | </div>
| |
| | | | |
| | | | |
| Line 4,840: |
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| | </div> | | </div> |
| | | | |
| - | | + | </div> |
| - | | + | </div> |
| | </section> | | </section> |
| | | | |
| Line 4,919: |
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| | <img src="https://static.igem.org/mediawiki/2014/7/79/Team_Berlin_tuberlin_logo.png" class="img-responsive grayscale"> | | <img src="https://static.igem.org/mediawiki/2014/7/79/Team_Berlin_tuberlin_logo.png" class="img-responsive grayscale"> |
| | </div> | | </div> |
| | + | |
| | + | <div class="col-sm-2 col-sm-offset-1 col-xs-3"> |
| | + | <img src="https://static.igem.org/mediawiki/parts/c/cb/Team_Berlin_florian_renner_logo.png" class="img-responsive grayscale"> |
| | + | </div> |
| | </div> | | </div> |
| | | | |
| Line 5,032: |
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| | }); // end window scroll | | }); // end window scroll |
| | }); // end section function | | }); // end section function |
| - |
| |
| - | $("#section-B").hide();
| |
| - | $("#section-C").hide();
| |
| - | $("#C-up").hide();
| |
| - | $("#B-up").hide();
| |
| - |
| |
| - | $("#show-B").click(function(){
| |
| - | $("#section-B").slideToggle();
| |
| - | $("#B-up").toggle();
| |
| - | $("#B-down").toggle();
| |
| - | });
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| - |
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| - | $("#show-C").click(function(){
| |
| - | $("#section-C").slideToggle();
| |
| - | $("#C-up").toggle();
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| - | $("#C-down").toggle();
| |
| - | });
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| | }); // close out script | | }); // close out script |
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