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Team:Bielefeld-CeBiTec/Results/rMFC
From 2014.igem.org
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We demonstrated that the knockout of C4 carboxylate antiporter <i>dcuB</i> was successful. | We demonstrated that the knockout of C4 carboxylate antiporter <i>dcuB</i> was successful. | ||
| - | Our constructed <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> strain shows no succinate export under anaerobic conditions. This demonstrates a successful knockout of the <i>dcuB</i> gene. Besides <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog® analysis</a> showed that there is no significant respiratory activity of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> in the presence of fumarate. | + | Our constructed <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> strain shows no succinate export under anaerobic conditions. This demonstrates a successful knockout of the <i>dcuB</i> gene. Besides <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog® analysis</a> showed that there is no significant respiratory activity of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> in the presence of fumarate. The electrobiochemical behavior of <i>E. coli</i> KRX with knocked out C4 carboxylate antiporter DcuB was tested in a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction#H-cellResults">H-cell reactor</a>. |
| - | The electrobiochemical behavior of <i>E. coli</i> KRX with knocked out C4 carboxylate antiporter DcuB was tested in a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction#H-cellResults">H-cell reactor</a>. | + | <br> |
| - | + | The second part deals with the investigation of fumarate reductase Frd (<a href="http://parts.igem.org/Part:BBa_K1465102">BBa_K1465102). Successful expression of the fumarate reductase Frd (<a href="http://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>) could be proven via <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/ElectronTransfer#SDSResults">SDS-PAGE</a>. | |
| - | The second part deals with the investigation of fumarate reductase Frd (<a href="http://parts.igem.org/Part:BBa_K1465102">BBa_K1465102). Successful expression of the fumarate reductase Frd (<a href="http://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>) could be proven via <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/ | + | Activity of the fumarate reductase displayed with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HPLC">HPLC analysis</a> of fumarate consumption and succinate production in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/ElectronTransfer#AnaerobFrdResults">anaerobic cultivation</a> of <i>E. coli</i> was shown with <a href="http://parts.igem.org/Part:BBa_K1465102">BBa_1465102</a>. Furthermore we investigated the fumarate reductase activity in different <i>E. coli</i> strains by phenotypic MicroArray (PM) analysis with a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/ElectronTransfer#BiologFrdResults">Biolog® system</a>. |
| - | Activity of the fumarate reductase displayed with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HPLC">HPLC analysis</a> of fumarate consumption and succinate production in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/ | + | |
<br> | <br> | ||
Revision as of 01:15, 18 October 2014
Module I - Reverse Microbial Fuel Cell (rMFC)
Our first module deals with the construction of an E. coli strain, which is able to accept electrons stimulating its metabolism. We characterized it in our electrobiochemical reactor system testing different mediators, electrode materials and reactor set-ups. Our genetical achievements could be devided in two parts.
In the first place we investigated the effect of the C4 carboxylate transporter DcuB knockout on E. coli KRX. Furthermore we showed the integration of the outer membrane porine OprF (BBa_K1172507) into the bacterial genome by replacing the gene of E. coli C4 carboxylate antiporter DcuB.
The funtionality of the genome integrated outer membrane porin OprF (BBa_K1172507) in E.coli KRX ΔdcuB::oprF was investigated with a NPN-Uptake-Assay.
We demonstrated that the knockout of C4 carboxylate antiporter dcuB was successful.
Our constructed E. coli KRX ΔdcuB::oprF strain shows no succinate export under anaerobic conditions. This demonstrates a successful knockout of the dcuB gene. Besides Biolog® analysis showed that there is no significant respiratory activity of E.coli KRX ΔdcuB::oprF in the presence of fumarate. The electrobiochemical behavior of E. coli KRX with knocked out C4 carboxylate antiporter DcuB was tested in a H-cell reactor.
The second part deals with the investigation of fumarate reductase Frd (BBa_K1465102). Successful expression of the fumarate reductase Frd (BBa_1465102) could be proven via SDS-PAGE.
Activity of the fumarate reductase displayed with HPLC analysis of fumarate consumption and succinate production in anaerobic cultivation of E. coli was shown with BBa_1465102. Furthermore we investigated the fumarate reductase activity in different E. coli strains by phenotypic MicroArray (PM) analysis with a Biolog® system.
