Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun

From 2014.igem.org

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<ul>
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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
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<ul>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of all four coding sequences for a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> </li>
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<ul>
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<li>Elongation time: 90 seconds</li>
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<li>Annealing temperature: 55°C</li>
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<li>Combinations of primer and template combinations are listed in the table below. Sequences for Gibson Assembly are introduced by the primers. Additionally the forward primers contain a RBS (BBa_B0034).</li>
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<table>
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<tr>
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<th>forward primer</th>
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<th>reverse primer</th>
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<th>template</th>
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</tr>
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<tr>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a></td>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS" target="_blank">rv_ilvC_alsS</a></td>
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<td><i>pSB1C3-alsS</i></td>
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</tr>
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<tr>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a></td>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a></td>
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<td><i>pSB1C3-ilvC</i></td>
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</tr>
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<tr>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a></td>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a></td>
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<td><i>pSB1C3-ilvD</i></td>
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</tr>
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<tr>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a></td>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a></td>
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<td><i>pSB1C3-kivD</i></td>
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</tr>
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<tr>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a></td>
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<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a></td>
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<td><i>pSB1C3-CFP</i></td>
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</tr>
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</table>
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<li>Products were analyzed by agarose gel electrophoresis</li>
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<ul>
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<li> PCR conditions need to be optimized do to low product quality and missing products</li>
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</ul>
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</ul>
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</ul>
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Revision as of 05:30, 27 August 2014


June

  • Transformation of electrocompotetent E.coli KRX cells:
    • K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
    • K539621: pSB1C3-ilvC (parts distribution from 2013, plate 1, 24F)
    • K539626: pSB1C3-ilvD (parts distribution from 2013, plate 1, 22J)
    • K539627: pSB1C3-alsS (parts distribution from 2013, plate 1, 24H)
  • Resulting colonies were selected to start liquid cultures:
    • 5 ml of LB in 20 ml reaction tube
    • incubation over night at 37°C and 250 rpm
  • Plasmid isolation by ThermoScientific MiniPrep
  • Sanger sequencing using VF and VR as sequencing primers
    • Identities of all parts were confirmed, but not whole sequence of the parts was covered
  • Glycerin stocks were created for all four strains:
    • E. coli KRX pSB1C3-alsS
    • E. coli KRX pSB1C3-ilvC
    • E. coli KRX pSB1C3-ilvD
    • E. coli KRX pSB1C3-kivD

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