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

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<li><b><i>prkA</i></b></li>
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        <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 <i>prkA</i>. 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>
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Revision as of 17:37, 16 October 2014


September







  • 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.


  • 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.


  • 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.

  • 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 ...



  • 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 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.

  • 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 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 ptac. 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.