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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Oct
From 2014.igem.org
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<li>The purified enzyme from His-Tag purification werde <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ProteinBufferExchangeAndDesalting" target="_blank">concentrated and desalted</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#ProteinPurification" target="_blank">Amicon Ultra-15</a> with 10kD cut-off</li> | <li>The purified enzyme from His-Tag purification werde <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ProteinBufferExchangeAndDesalting" target="_blank">concentrated and desalted</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#ProteinPurification" target="_blank">Amicon Ultra-15</a> with 10kD cut-off</li> | ||
</ul> | </ul> | ||
| + | <li>Additionally we tried to establish an <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RuBisCOactivityassay" target="_blank">enzyme assay</a> for our own construct of the <i>glpX</i> (SBPase). | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<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:120px; text-align:center"> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/7/77/Bielefeld_CeBiTec_2014-10-17_prkA_Kontrollverdau_10_09.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/77/Bielefeld_CeBiTec_2014-10-17_prkA_Kontrollverdau_10_09.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> | ||
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<li><b>T7_Hneap</b></li> | <li><b>T7_Hneap</b></li> | ||
<ul> | <ul> | ||
| - | <li> The expression of the construct T7_Hneap was verified via <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a>. <li> 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, | + | <li> The expression of the construct T7_Hneap was verified via <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a>. <li> 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 0,1 % rhamnose (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> |
</ul> | </ul> | ||
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<div class="content" style="margin-right:10%; margin-left:10%"> | <div class="content" style="margin-right:10%; margin-left:10%"> | ||
<ul> | <ul> | ||
| - | <li><b>p<sub>tac</sub> | + | <li><b>p<sub>tac</sub>_glpX</b></li> |
<ul> | <ul> | ||
<li>We made a cultivation with our construct pSB1C3_p<sub>tac</sub>_glpX in xylose to see if there is a difference between this cultivation and the one with glucose and also between induced and not induced. As a reference we used the <i>E. coli</i> wildtype. We used 30 ml in 250 ml flasks a 37°C and 250 rpm. The induction was with IPTG at on OD<sub>600</sub> of 0.7. The cultivation lasted 15 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> | <li>We made a cultivation with our construct pSB1C3_p<sub>tac</sub>_glpX in xylose to see if there is a difference between this cultivation and the one with glucose and also between induced and not induced. As a reference we used the <i>E. coli</i> wildtype. We used 30 ml in 250 ml flasks a 37°C and 250 rpm. The induction was with IPTG at on OD<sub>600</sub> of 0.7. The cultivation lasted 15 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> | ||
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</ul> | </ul> | ||
<br> | <br> | ||
| + | |||
| + | <br> | ||
| + | <ul> | ||
| + | <li><b><i>glpX</i></b></li> | ||
| + | <ul> | ||
| + | <li>Again we tried to establish our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RuBisCOactivityassay" target="_blank">enzyme assay</a> for our own construct of the <i>glpX</i> (SBPase). | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<ul> | <ul> | ||
<li><b>T7_sap_csoS1-4_GFP and T7_csoS1-4_GFP</b></li> | <li><b>T7_sap_csoS1-4_GFP and T7_csoS1-4_GFP</b></li> | ||
Latest revision as of 00:37, 18 October 2014
 |
October |
- tkt
- This week we wanted to do the enzyme assay for the SBPase.
- The purified enzyme from His-Tag purification werde concentrated and desalted with Amicon Ultra-15 with 10kD cut-off
- fba
- This week we wanted to do the enzyme assay for the SBPase.
- The purified enzyme from His-Tag purification werde concentrated and desalted with Amicon Ultra-15 with 10kD cut-off
- glpX
- This week we wanted to do the enzyme assay for the SBPase.
- The purified enzyme from His-Tag purification werde concentrated and desalted with Amicon Ultra-15 with 10kD cut-off
- Additionally we tried to establish an enzyme assay for our own construct of the glpX (SBPase).
- prkA and ptac_Hneap
- We tried to bring the prkA with RBS behind our construct ptac_Hneap but it was not successful.
- prkA
- We tried to bring our prkA with RBS in pSB1C3.
- BioBrick Assembly (Suffix)
- Transformation with chemocompetent cells
- Plasmid isolation of [Construct]
- Restriction digestion with PstI and EcoRI
- Bands as expected (~2000 bp and ~1100 bp)
- ptac_glpX
- We made a cultivation with our construct pSB1C3_ptac_glpX in xylose and glucose to see if there is a difference between the two media and also between induced and not induced. As a reference we used the E. coli wildtype. Because the culture in xylose did not grow we only could measure the OD600 of the glucose cultures. We used 30 ml in 250 ml flasks a 37°C and 250 rpm. The induction was with IPTG at on OD600 of 0.7. The cultivation lasted 12 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.
- T7_Hneap
- The expression of the construct T7_Hneap was verified via SDS-Page.
- Cultivation was carried out using the method of Cultivation for Expression of recombinant proteins. Protein expression was induced with 0,1 % rhamnose (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.
- ptac_Hneap
- The expression of the construct T7_Hneap was verified via SDS-Page.
- 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 T7_Hneap induced with 0.5 mM IPTG.
Proteinexpression of ptac_Hneap induced with 0.5 mM IPTG.
- ptac_glpX
- We made a cultivation with our construct pSB1C3_ptac_glpX in xylose to see if there is a difference between this cultivation and the one with glucose and also between induced and not induced. As a reference we used the E. coli wildtype. We used 30 ml in 250 ml flasks a 37°C and 250 rpm. The induction was with IPTG at on OD600 of 0.7. The cultivation lasted 15 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.
- glpX
- Again we tried to establish our enzyme assay for our own construct of the glpX (SBPase).
- T7_sap_csoS1-4_GFP and T7_csoS1-4_GFP
- We tried to prove that the carboxysome we built can form the micro compartiment for the CO2 fixation. Therefore we measured the GFP fluorescence signal with the GloMax® Discover Multimode-Reader of Promega. We took 1 ml of the culture, washed it with 1 ml 1x PBS buffer and resuspend it in 600 µl 1x PBS buffer. For the measurement we used 200 µl for each technical replica in a 96 plate (black). The given protocol for GFP fluorescence was used.
Additionally we examined the samples under a microscope.
