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

From 2014.igem.org

(Difference between revisions)
Line 440: Line 440:
             <li>We tried to finish our carboxysome with and without <i>csoS1D</i> 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.</li>
             <li>We tried to finish our carboxysome with and without <i>csoS1D</i> 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.</li>
           </ul>
           </ul>
-
</ul>
+
 
 +
 
 +
 
 +
<br>
 +
 
 +
<li><b><i>Purification of the carboxysome</b></i></li>
 +
                <ul>
 +
                    <li>The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression 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 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification. </li>
 +
                   
 +
&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
 +
                    </ul>
 +
                   
 +
                </ul>
           </div>
           </div>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
 +
 +
 +
 +
 +
 +
 +
 +

Revision as of 15:35, 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 litre. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling.
    • → 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
    • The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression 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 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification.
    • → The purification was not succesful, as you could not recognize a visible band in the gradient.


  • csoS1-4_GFP and T7_sap

  • 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 ...



  • 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.