Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep

From 2014.igem.org

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Line 128: Line 128:
<li><b><i>csoS1-4_GFP</i> and T7</b></li>
<li><b><i>csoS1-4_GFP</i> and T7</b></li>
                 <ul>
                 <ul>
-
                   <li>We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.</li>
+
                   <li>We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promoter.</li>
                   <ul>
                   <ul>
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
Line 241: Line 241:
<li><b><i>Hneap</i> and <i>T7</i></b></li>
<li><b><i>Hneap</i> and <i>T7</i></b></li>
          <ul>
          <ul>
-
    <li>This week we tried to assemble the <i>T7</i> promotor with the <i>Hneap</i>.</li>  
+
    <li>This week we tried to assemble the <i>T7</i> promoter with the <i>Hneap</i>.</li>  
                     <ul>
                     <ul>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
Line 289: Line 289:
<li><b><i>glpX</i> and p<sub>tac</sub> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465229" target="_blank">(BBa_K1465229)</a></b></li>
<li><b><i>glpX</i> and p<sub>tac</sub> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465229" target="_blank">(BBa_K1465229)</a></b></li>
                 <ul>
                 <ul>
-
                   <li>This week we tried to bring <i>glpX</i> in the pSB1C3 backbone under the control of the p<sub>tac</sub> promotor.</li>
+
                   <li>This week we tried to bring <i>glpX</i> in the pSB1C3 backbone under the control of the p<sub>tac</sub> promoter.</li>
                   <ul>
                   <ul>
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
Line 320: Line 320:
<ul>
<ul>
<li>Bands as expected (~2000 bp and ~2200 bp)</li>
<li>Bands as expected (~2000 bp and ~2200 bp)</li>
 +
<div class="element" style="height:350px; width:150px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/6/64/Bielefeld_CeBiTec_2014-10-17_ptac_glpX_Kontrollverdau_09_09.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/6/64/Bielefeld_CeBiTec_2014-10-17_ptac_glpX_Kontrollverdau_09_09.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
</ul>
</ul>
</ul>
</ul>
Line 328: Line 332:
<br>
<br>
-
<li><b><i>Purification of the carboxysome</b></i></li>
+
<li><b>Purification of the carboxysome</b></li>
                 <ul>
                 <ul>
                     <li>Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. The culture volumen was upscaled to 1 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min) to obtain a better concentration and seperation. </li>
                     <li>Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. The culture volumen was upscaled to 1 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min) to obtain a better concentration and seperation. </li>
Line 445: Line 449:
<br>
<br>
-
<li><b><i>Purification of the carboxysome</b></i></li>
+
<li><b>Purification of the carboxysome</b></li>
                 <ul>
                 <ul>
                     <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer.  </li>
                     <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer.  </li>
Line 481: Line 485:
<li><b>T7 and <i>prkA</i></b></li>
<li><b>T7 and <i>prkA</i></b></li>
               <ul>
               <ul>
-
               <li>This week we wanted to bring the <i>prkA</i> under the control of the T7 promotor.</li>
+
               <li>This week we wanted to bring the <i>prkA</i> under the control of the T7 promoter.</li>
               <ul>
               <ul>
                 <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
                 <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
Line 514: Line 518:
<ul>
<ul>
<li>Bands as expected (~2000 bp and ~1100 bp)</li>
<li>Bands as expected (~2000 bp and ~1100 bp)</li>
 +
<div class="element" style="height:350px; width:140px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/d/dc/Bielefeld_CeBiTec_2014-10-17_T7_prkA_Kontrollverdau_09_26.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/dc/Bielefeld_CeBiTec_2014-10-17_T7_prkA_Kontrollverdau_09_26.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
 +
                    </div>
 +
</ul>
</ul>
</ul>
</ul>
Line 526: Line 534:
<li><b>T7 and <i>sap</i></b></li>
<li><b>T7 and <i>sap</i></b></li>
               <ul>
               <ul>
-
               <li>We tried to bring the <i>sap</i> under the control of the T7 promotor.</li>
+
               <li>We tried to bring the <i>sap</i> under the control of the T7 promoter.</li>
               <ul>
               <ul>
                 <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
                 <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
Line 569: Line 577:
<li><b><i>csoS1-4_GFP</i> and T7_sap</b></li>
<li><b><i>csoS1-4_GFP</i> and T7_sap</b></li>
<ul>
<ul>
-
         <li>We tried to assemble our GFP construct with the shell associated protein <i>sap</i> and the T7 promotor.</li>   
+
         <li>We tried to assemble our GFP construct with the shell associated protein <i>sap</i> and the T7 promoter.</li>   
         <ul>
         <ul>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Prefix)</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Prefix)</li>
Line 597: Line 605:
<br>
<br>
-
<li><b><i>Purification of the carboxysome</b></i></li>
+
<li><b>Purification of the carboxysome</b></li>
                 <ul>
                 <ul>
                     <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. With the resuspended pellet two different ultracentrifugations were done. One centrifugation for 30 min, one further centrifugation for 60 min.  </li>
                     <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. With the resuspended pellet two different ultracentrifugations were done. One centrifugation for 30 min, one further centrifugation for 60 min.  </li>
Line 644: Line 652:
<ul>
<ul>
<li>Bands as expected (~2000 bp and ~4600 bp)</li>
<li>Bands as expected (~2000 bp and ~4600 bp)</li>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/c/c2/Bielefeld_CeBiTec_2014-10-17_T7_sap_csoS1-4_gfp_Kontrollverdau_10_02.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c2/Bielefeld_CeBiTec_2014-10-17_T7_sap_csoS1-4_gfp_Kontrollverdau_10_02.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.neb.com/products/n3232-1-kb-dna-ladder">1 kb DNA Ladder from NEB</a>. </font>
 +
                    </div>
 +
</ul>
</ul>
</ul>
</ul>
Line 735: Line 747:
<ul>
<ul>
<li>Bands as expected (~2000 bp and ~1000 bp)</li>
<li>Bands as expected (~2000 bp and ~1000 bp)</li>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/3b/Bielefeld_CeBiTec_2014-10-17_glpX_Kontrollverdau_10_06.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/3b/Bielefeld_CeBiTec_2014-10-17_glpX_Kontrollverdau_10_06.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.neb.com/products/n3232-1-kb-dna-ladder">1 kb DNA Ladder from NEB</a>. </font>
 +
                    </div>
</ul>
</ul>
</ul>
</ul>
-
<br>
 
-
<li>Additionally we tried to bring <i>glpX</i> under the control of the T7 promotor.</li>
 
-
 
-
<ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
 
-
<ul>
 
-
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 
-
<ul>
 
-
<li>pSB1A2_T7</li>
 
-
</ul>
 
-
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li>
 
-
<ul>
 
-
<li><i>glpX</i></li>
 
-
</ul>
 
-
</ul>
 
-
</ul>
 
-
<ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 
-
</ul>
 
-
<ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)
 
-
            </li>
 
-
<ul>
 
-
<li>Annealing temperature: ... °C</li>
 
-
<li>Bands (not) as expected (~... bp)</li>
 
-
</ul>
 
-
</ul>
 
-
 
-
 
-
                </ul>
 
-
              </ul>
 
<br>
<br>
Line 779: Line 763:
</ul>
</ul>
<ul>
<ul>
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix/Prefix)</li>
+
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
<ul>
<ul>
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
Line 804: Line 788:
<ul>
<ul>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_prkA</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_prkA</li>
-
</ul>
 
-
<ul>
 
-
<li><font color="red"><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>[Enzyme]</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>[Enzyme]</i>I</a> </li>
 
-
<ul>
 
-
<li>Bands as expected (~ bp)</li>
 
-
</ul></font>
 
</ul>
</ul>
Line 816: Line 794:
<br>
<br>
 +
 +
 +
 +
 +
 +
 +
 +
<ul>
 +
<li><b><i>prkA</i></b></li>
 +
      <ul>
 +
        <li>After recognation of the fact, that our pSB1C_prkA construct did not have the desired RBS, we wanted to see whether there is protein expression after induction of the construct p<sub>tac</sub>_prkA. Cultivation was carried out using the method of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">Cultivation for Expression of recombinant proteins</a>. Protein expression was induced with 0,5 mM IPTG (final concentration). To verify the expression of the RuBisCo through <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS-PAGE </a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Matrix-assistedLaserDesorption/Ionization%E2%80%93Timeofflight%20%28MALDI-TOF%29" target="_blank">MALDI-TOF</a>, samples were generated using the protocol for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">Fast Cell Lysis for SDS-PAGE. </a></</li>
 +
 +
<center>
 +
<div class="element" style="width:350px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" width="350"></a><br>
 +
<font size="2">Proteinexpression of p<sub>tac</sub>_prkA induced with 0.5 mM IPTG. </font>
 +
                    </div> </center>
 +
 +
 +
       
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
<ul>
<li><b>T7_sRNA:pfkA and p<sub>tac</sub>_sRNA:pfkA</b></li>
<li><b>T7_sRNA:pfkA and p<sub>tac</sub>_sRNA:pfkA</b></li>
       <ul>
       <ul>
-
         <li>We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of <i>E. coli</i>. The cultivations were made in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C. The induction was with rhamnose for the T7 promotor and IPTG for the p<sub>tac</sub>. It lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for HPLC analysis that followed.</li>
+
         <li>We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of <i>E. coli</i>. The cultivations were arranged in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C and 250 rpm. The induction was with rhamnose for the T7 promoter and IPTG for the p<sub>tac</sub> at an OD<sub>600</sub> value of 0.6. The cultivation lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HPLC" target="_blank">HPLC</a> analysis that followed.</li>
</ul>
</ul>
</ul>
</ul>

Latest revision as of 00:35, 18 October 2014


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07






Week 2     09/08 - 09/14
Week 2     09/08 - 09/14
  • glpX and ptac (BBa_K1465229)

  • Purification of the carboxysome
    • Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. The culture volumen was upscaled to 1 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min) to obtain a better concentration and seperation.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 3     09/15 - 09/21
Week 3     09/15 - 09/21


  • can_csoS1-4 respectively can_csoS1-4_csoS1D and sap
    • We tried to finish our carboxysome with and without csoS1D but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.

  • Purification of the carboxysome
    • Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28


  • csoS1-4_GFP and T7_sap

  • Purification of the carboxysome
    • Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. With the resuspended pellet two different ultracentrifugations were done. One centrifugation for 30 min, one further centrifugation for 60 min.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 5     09/29 - 10/05
Week 5 09/29 - 10/05

  • tkt
    • This week we wanted to purify the enzyme of tkt for the SBPase assay.
      • Cultivation of pet16b_tkt in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~100kD
      • His-Tag purification of tkt
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

  • fba
    • This week we wanted to purify the enzyme of fba for the SBPase assay.
      • Cultivation of pet16b_fba in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~40kD
      • His-Tag purification of fba
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

  • glpX (BBa_K1465228 (pSB1C3_glpX))
    • This week we wanted to purify the enzyme of for the SBPase assay.
      • Cultivation of pet16b_glpX in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~38kD
      • His-Tag purification of glpX
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

    • We also tried to bring glpX in the right vector pSB1C3.


      • prkA
        • After recognation of the fact, that our pSB1C_prkA construct did not have the desired RBS, we wanted to see whether there is protein expression after induction of the construct ptac_prkA. Cultivation was carried out using the method of Cultivation for Expression of recombinant proteins. Protein expression was induced with 0,5 mM IPTG (final concentration). To verify the expression of the RuBisCo through SDS-PAGE and MALDI-TOF, samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE.

          Proteinexpression of ptac_prkA induced with 0.5 mM IPTG.

      • T7_sRNA:pfkA and ptac_sRNA:pfkA
        • We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of E. coli. The cultivations were arranged in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C and 250 rpm. The induction was with rhamnose for the T7 promoter and IPTG for the ptac at an OD600 value of 0.6. The cultivation lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for HPLC analysis that followed.

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