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

From 2014.igem.org

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<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
<ul>
<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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<li>This week 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>
                     <ul>
                     <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 amplified many parts for different combination possibilities in the Gibson Assembly:  
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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 amplified five parts for two combination possibilities in the Gibson Assembly:  
<ol><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> on pSB1C3_RFP <br>&#8594; pSB1C3 with ~ 2200 bp</li>
<ol><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> on pSB1C3_RFP <br>&#8594; pSB1C3 with ~ 2200 bp</li>
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<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
</ul>
</ul>
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<ul>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with the previous PCR products: <ul>
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<li>1., 2. and 3.</li><li>1., 2., 4. and 5. </li>
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</ul></li>
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</ul>
</ul>
</ul>

Revision as of 20:47, 15 October 2014


September

  • 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


  • 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
  • pSB1C3_alsS_ilvC_ilvD_kivD
    • This week we used old restiction digestions for new ligations without success.