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

From 2014.igem.org

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<ul>
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<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
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<ul>
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<li>This week we used old restiction digestions for new ligations without success.</li>
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</ul>
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</ul>
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<ul>
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<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
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<ul>
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<li>This week we the new primers <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> arrived. We tried to remove the illegale restriction side.</li>
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                    <ul>
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<li>We tried different<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>s to increase the chance to get a correct BioBrick, therefore we amplify many parts for different combination possibilities in the Gibson Assembly:
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<ul><li>  (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li></ul>
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<ul><li>  (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primerfw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>)</li></ul>
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<ul><li>  (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a>)</li></ul>
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<ul><li>  (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a>)</li></ul>
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<ul><li>  (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li></ul></li>
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<ul>
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<li>Annealing temperature: 65 &deg;C</li>
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<li>Bands (not) as expected (~... bp)</li>
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</ul>
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</ul>
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</ul>
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</ul>
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Revision as of 19:23, 15 October 2014


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07
  • T7_alsS_ilvC_ilvD_kivD
    • This week we tried to verify the positive cutures that were identified last week by a restriction digest
  • pSB1K3_alsS_ilvC_ilcD_kivD_adhA

Week 2     09/08 - 09/14
Week 2     09/08 - 09/14

Week 3     09/15 - 09/21
Week 3     09/15 - 09/21
  • pSB1C3_alsS_ilvC_ilvD_kivD_adhA
    • This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
      • NEB BioBrick Assembly
        • Upstream part pSB1K3_alsS_ilvC_ilvD_kivD (digested with EcoRI, SpeI)
          • alsS_ilvC_ilvD_kivD
        • Downstream part pSB1K3_adhA(digested with XbaI, PstI)
          • adhA
        • Destination part pSB1C3_RFP (digested with EcoRI, PstI)
          • alsS_ilvC_ilvD_kivD
      • Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_alsS_ilvC_ilvD_kivD has an illigal restriction side.
        Because of the integration of RBS's between all genes in the alsS_ilvC_ilvD_kivD-Plasmid, a restriction side between alsS and a RBS occurs.
        New primers were ordered: rv_ilvC_alsS-new and fw_alsS_ilvC-new
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28
  • pSB1C3_alsS_ilvC_ilvD_kivD
    • This week we used old restiction digestions for new ligations without success.

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