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

From 2014.igem.org

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<ul>
<ul>
<li>This week we arranged the complete <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction">H-cell reactor </a> for the first time to start first trials. Therefore we prepare the<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank"> H-cell buffers</a> and the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Nafion%C2%AE%20membrane" target="_blank">Nafion® membrane</a> which ensures the division of the two compartments.</li>
<li>This week we arranged the complete <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction">H-cell reactor </a> for the first time to start first trials. Therefore we prepare the<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank"> H-cell buffers</a> and the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Nafion%C2%AE%20membrane" target="_blank">Nafion® membrane</a> which ensures the division of the two compartments.</li>
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</ul>
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    <ul><li>Additionally we started to cultivate in the H-cell reactor to evaluate the growth of <i>E. coli</i> under the current intesity which is according to the literature needed to reduce neutral red and bromphenol blue.</li>                 
</ul>
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</ul>  
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of all constructs with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of all constructs with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
<ul>
<ul>
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<li>Bands (not) as expected (~... bp)</li>
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<li>Bands as expected (~3300 bp and ~2070 bp)</li>
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             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
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<ul>
 
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<li><b><i>frd (E. coli) </i></b></li>
 
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<ul>
 
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<li> This week we removed the first illegal restriction site (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> at 144 bp)
 
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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> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#cut1" target="_blank">frd_cut1</a>
 
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<ul>
 
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<li>Annealing temperature: 55 &deg;C</li>
 
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<li>Bands as expected (~... bp)</li>
 
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</ul>
 
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</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> of PCR products and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Dpn</i>I</a> digestion to remove the template
 
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</ul>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 
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</ul>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> for a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> afterwards </li>
 
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<ul>
<li><b><i>ccm</i></b></li>
<li><b><i>ccm</i></b></li>
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<br>
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<ul>
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<li><b>Electrobiochemical reactor system</b></li>
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<ul>
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<li> This week we performed several cyclic voltammetric measurements to characterize different electrode materials and mediators. Therefore the system was purged with nitrogen to remove any oxygen.</li>
 +
<li> Measurements were performed under the following conditions and the the following experimental setup</li>
 +
<ul>
 +
<li> Cathode space: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank">cathode buffer</a> or <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium" target="_blank">M9 medium</a></li>
 +
<li> Anode space: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#H-cell%20buffer" target="_blank">anode buffer</a> 
 +
<li> Working electrode: platinic plate or graphite electrode</li>
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<li> Counter electrode: platinic wire </li>
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<li> Reference electrode: Ag/AgCl- electrode</li>
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<li> Scan rate: 10 mV/s</li>
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<li> Scan Limit: was varied among the different measurements</li>
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<li> Cycle number: 3, 10 or 500</li>
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<li> Mediators: neutral red or bromphenol blue</li>
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<li> Temperature: 37&deg;C</li>
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</ul>
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<li>induction was performed by adding 0,1% Rhamnose</li>
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<li>Induction was performed by adding 0,1% Rhamnose</li>
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics
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</ul>
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<ul>
<ul>
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<li>induction was performed by adding 0,1% Rhamnose</li>
+
<li>Induction was performed by adding 0,1% Rhamnose</li>
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics
<li>To document the growth curve and the consumption of substrate daily samples were taken for OD<sub>600</sub> measurement and HPLC analytics

Latest revision as of 19:46, 15 October 2014


September



  • GSU 3274
    • This week we assembled GSU 3274 with different promotors

  • Electrobiochemical reactor system
    • Additionally we started to cultivate in the H-cell reactor to evaluate the growth of E. coli under the current intesity which is according to the literature needed to reduce neutral red and bromphenol blue.



  • Electrobiochemical reactor system
    • This week we performed several cyclic voltammetric measurements to characterize different electrode materials and mediators. Therefore the system was purged with nitrogen to remove any oxygen.
    • Measurements were performed under the following conditions and the the following experimental setup
      • Cathode space: cathode buffer or M9 medium
      • Anode space: anode buffer
      • Working electrode: platinic plate or graphite electrode
      • Counter electrode: platinic wire
      • Reference electrode: Ag/AgCl- electrode
      • Scan rate: 10 mV/s
      • Scan Limit: was varied among the different measurements
      • Cycle number: 3, 10 or 500
      • Mediators: neutral red or bromphenol blue
      • Temperature: 37°C

  • Deletion of dcuB and integration of oprF into chromosome

  • FumBCD

    • anaerobic cultivation for characterization of pSB1C3_T7_frd
      • We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
        • Strains
          • KRX wildtype B0015
          • KRX wildtype with pSB1C3_T7_frd
          • JW0506-1
          • JW0506-1 with pSB1C3_T7_frd
        • Media
          • M9 with xylose (50 mM)
          • M9 with xylose (50 mM) and fumarate (50 mM)
          • M9 with fumarate (50 mM)
        • Induction was performed by adding 0,1% Rhamnose
        • To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics

    • anaerobic cultivation for characterization of the antiporter δdcuB
      • We tested all combinations of the following media and strains for the characterization of pSB1C3_T7_frd
        • Strains
          • KRX wildtype B0015
          • KRX wildtype with pSB1C3_T7_frd
          • KRX ΔdcuB::oprF
          • KRX ΔdcuB::oprF with pSB1C3_T7_frd
          • JW4084-1
          • JW4084-1 with pSB1C3_T7_frd
          • JW0506-1
          • JW0506-1 with pSB1C3_T7_frd
        • Media
          • M9 with xylose (50 mM)
          • M9 with xylose (50 mM) and fumarate (50 mM)
          • M9 with fumarate (50 mM)
        • Induction was performed by adding 0,1% Rhamnose
        • To document the growth curve and the consumption of substrate daily samples were taken for OD600 measurement and HPLC analytics