|
|
| (10 intermediate revisions by the same user not shown) |
| Line 1: |
Line 1: |
| | __NOTOC__ | | __NOTOC__ |
| | <partinfo>BBa_K1465109 short</partinfo> | | <partinfo>BBa_K1465109 short</partinfo> |
| − | | + | ---- |
| − | dcuB fwd | + | <html> |
| | + | <p align="justify"> |
| | + | Primer dcuB fwd (<a href="https://parts.igem.org/Part:BBa_K1465109">BBa_K1465109</a>) is part of the construction of an electrophilic <i>E. coli</i> strain, which metabolism could be increased by growing under influence of electric power. It is part of a primer collection containing <a href="https://parts.igem.org/Part:BBa_K1465107">BBa_K1465107</a>, <a href="https://parts.igem.org/Part:BBa_K1465108">BBa_K1465108</a>, <a href="https://parts.igem.org/Part:BBa_K1465109">BBa_K1465109</a> and <a href="https://parts.igem.org/Part:BBa_K1465110">BBa_K1465110</a>. The aim is to create a deletion cassette for knocking out C4 carboxylate transporter DcuB and insert outer membrane porine OprF (<a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>) into <i>E. coli</i> chromosome in the same step. |
| | + | <br> |
| | + | </html> |
| | | | |
| | ===Usage and Biology=== | | ===Usage and Biology=== |
| − | | + | ---- |
| | + | <html> |
| | <h4>C4 Carboxylate Antiporter DcuB</h4> | | <h4>C4 Carboxylate Antiporter DcuB</h4> |
| | + | <p align="justify"> |
| | Under anaerobic conditions <i>E. coli</i> cells use different alternative electron acceptors instead of oxygen. Partially the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential less-oxidizing substances for bacteria to release their electrons, for example anorganic compounds like nitrate (NO<sub>3</sub><sup>-</sup>) or sulfate (SO<sub>4</sub><sup>2-</sup>).(<a href="#Gottschalk1986">Gottschalk <i>et al.</i>, 1986</a>) | | Under anaerobic conditions <i>E. coli</i> cells use different alternative electron acceptors instead of oxygen. Partially the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential less-oxidizing substances for bacteria to release their electrons, for example anorganic compounds like nitrate (NO<sub>3</sub><sup>-</sup>) or sulfate (SO<sub>4</sub><sup>2-</sup>).(<a href="#Gottschalk1986">Gottschalk <i>et al.</i>, 1986</a>) |
| | Fumarate respiration leads to succinate excretion through the C4 carboxylate transporter DcuB. It is an antiporter which exchanges fumarate against succinate under anaerobic conditions. Under aerobic condition there is usually no succinate release observed. In connection to the carbon dioxide fixation in our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. So in case of oxygen limiting conditions, there could occured partial fumarate respiration in <i>E. coli</i>. Besides there was shown activity of DcuB antiporter in the presence of high fumarate concentrations (<a href="#Janausch2001">Janausch, 2001</a>). To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our <i>E. coli</i> strain. <br> | | Fumarate respiration leads to succinate excretion through the C4 carboxylate transporter DcuB. It is an antiporter which exchanges fumarate against succinate under anaerobic conditions. Under aerobic condition there is usually no succinate release observed. In connection to the carbon dioxide fixation in our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. So in case of oxygen limiting conditions, there could occured partial fumarate respiration in <i>E. coli</i>. Besides there was shown activity of DcuB antiporter in the presence of high fumarate concentrations (<a href="#Janausch2001">Janausch, 2001</a>). To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our <i>E. coli</i> strain. <br> |
| − |
| + | We planned a targeted knockout of the <i>dcuB</i> gene in <i>E. coli</i> KRX using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf">Genebridge Red/ET-System</a>. In the same step we are going to integrate the outer membrane porine OprF (<a href="https://parts.igem.org/Part:BBa_K1172507">BBa_K1172507</a>) into the bacterial chromosome under controll of a constitutive promotor (<a href="https://parts.igem.org/Part:BBa_J23104">BBa_J23104</a>). This ensure the permeability of the outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.<br> |
| − | We planned a targeted knockout of the <i>dcuB</i> gene in <i>E. coli</i> KRX using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf " target="_blank">Genebridge Red/ET-System</a>. In the same step we are going to integrate the outer membrane porine OprF (<a href="https://parts.igem.org/Part:BBa_K1172507">BBa_K1172507</a>) into the bacterial chromosome under controll of a constitutive promotor (<a href="https://parts.igem.org/Part:BBa_J23104">BBa_J23104</a>). This ensure the permeability of the outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.<br> | + | </p> |
| − | | + | </html> |
| − | | + | |
| | | | |
| | + | ===Sequence and Features=== |
| | <!-- --> | | <!-- --> |
| − | <span class='h3bb'>Sequence and Features</span>
| |
| | <partinfo>BBa_K1465109 SequenceAndFeatures</partinfo> | | <partinfo>BBa_K1465109 SequenceAndFeatures</partinfo> |
| | | | |
| Line 26: |
Line 31: |
| | ===Results=== | | ===Results=== |
| | <html> | | <html> |
| − | <h4>Construction of an electrophilic <i>E. coli</i> strain</h4>
| + | For detailed information see <a href="https://parts.igem.org/Part:BBa_K1465110">BBa_K1465110</a>. |
| − | <p>
| + | |
| − | We construct an <i>E. coli</i> strain, which is able to accept electrons stimulating its metabolism.
| + | |
| − | Direct electron transfer in bacteria is a very complex and not completely cleared up so far. Due to this we focused on the indirect electron transfer via a mediator, which is reduced at the electrode in a electrobiochemical reactor and would be reoxidized again by bacterial cells.(<a href="#Park1999">Park <i>et al.</i>, 1999</a>) In gram-negative bacteria <i>E. coli</i> there are two membranes with a periplasmatic space between them, which has to be overcome in the course of the electron transfer.<br>
| + | |
| − | We worked on three different mediators: neutral red, bromphenol blue and cytochromes. Additionally we constructed an electropholic <i>E. coli</i> strain, which shows an increased metabolic activity growing with electric power, which has been proven in our <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction#H-cellResults">H-cell reactor</a> (<a href="#Park1999">Park <i>et al.</i>, 1999</a>).<br>
| + | |
| − | The electron transfer system consists of different steps. First of all the reduced mediator has to cross the outer membrane of the <i>E. coli</i> cell. For that we used the outer membrane porine OprF (<a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>) provided by the <a href="http://2013.igem.org/Team:Bielefeld-Germany/Project/Porins">iGEM Team Bielefeld-Germany 2013</a>. Crossing the periplasmatic space, the mediator adsorb at the inner membrane of the <i>E. coli</i> cell. The mediator functions as an electron donor for the over expressed fumarate reductase. In this step succinate is produced in the cytoplasm. Reduction of fumarate into succinate creates a loop into the citric acid cycle, because succinate would be reoxidized again by the succiante dehydrogenase. Succinate excretion is avoid, because of the knockout of the C4 carboxylate transporter DcuB. In the reaction of succinate dehydrogenase electrons are transferred to FAD<sup>+</sup> generating FADH<sub>2</sub>, which enter the electron transport chain. The electron transport facilitates proton translocation over the inner bacterial membrane. The proton motoric force is used by ATP synthase. Generated ATP and reductive power in the bacterial cell leads to an increasing metabolic activity.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | | + | |
| − | <h4><i>E. coli</i> KRX ΔdcuB::oprF strain</h4>
| + | |
| − | <p>
| + | |
| − | We investigated the effect of the C4 carboxylate transporter DcuB knockout on <i>E. coli</i> KRX. Furthermore we showed the integration of the outer membrane porine OprF (<a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>) into the bacterial genome by replacing the gene of E. coli C4 carboxylate antiporter DcuB. So we did knockout and insertion in a one-step process.
| + | |
| − | The successful knockout of the DcuB antiporter and simultaneous insertion of <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a> was shown with PCR analysis, DNA sequencing and phenotypic investigation via <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog analysis</a> and <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> in M9 minimal media with fumarate supplemented. Substrates and products were analyzed by HPLC.
| + | |
| − | The electrobiochemical behavior of <i>E. coli</i> KRX with knocked out C4 carboxylate antiporter DcuB was tested in a <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/Construction#H-cellResults">H-cell reactor</a>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h4>Preparation of knockout deletion cassette</h4>
| + | |
| − | <p>
| + | |
| − | We decide to knock out the C4 carboxylate transporter DcuB to prevent succinate export. Besides we inserted the outer membrane porines OprF (<a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>) into the genome. Both could be realized with the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System</a>.
| + | |
| − | For simultaneous knockout and insertion a deletion cassette had to be designed and established. We used overlap extension PCR to amplify the complete deletion cassette (<a href="#Bryksin&Matsumura2010">Bryksin & Matsumura, 2010</a>). We used kanamycin as an antibiotic selective marker amplified from the plasmid Flp705 using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System protocoll</a>.
| + | |
| − | We designed the primers (<a href="https://parts.igem.org/Part:BBa_K1465107">BBa_K1465107</a>, <a href="https://parts.igem.org/Part:BBa_K1465108">BBa_K1465108</a>, <a href="https://parts.igem.org/Part:BBa_K1465109">BBa_K1465109</a>, <a href="https://parts.igem.org/Part:BBa_K1465110">BBa_K1465110</a> ) with complementary 5´extensions for both fragments to connect them in overlap extension PCR. The amplified deletion cassette has homologous sites for recombination with the <i>dcuB</i> gene in the <i>E. coli</i> KRX genome. | + | |
| − | </p>
| + | |
| − | | + | |
| − | | + | |
| − | <h4>Verification of knockout and deletion cassette</h4>
| + | |
| − | <p>
| + | |
| − | <table align="left" cellspacing=10" style="background-color:transparent; float:left">
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; float:left">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/7/7b/Bielefeld-CeBiTec_14-10-17_cPCR_Deletion.png" target="_blank" "text-align:left;"><img src="https://static.igem.org/mediawiki/2014/7/7b/Bielefeld-CeBiTec_14-10-17_cPCR_Deletion.png" width="75px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 1</b>:
| + | |
| − | Results of the colony PCR for<br>
| + | |
| − | analysis of <i>dcuB</i> genome area<br>
| + | |
| − | analyzed via agarose gelelectrophoresis. <br>
| + | |
| − | <i>E. coli</i> KRX wildtype band <br>
| + | |
| − | is shown on the rigth of about 2391 bp. <br>
| + | |
| − | <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> band is shown <br>
| + | |
| − | on the left at 4046 bp.</font <br>
| + | |
| − | </div>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | | + | |
| − | | + | |
| − | The gene of the C4 carboxylate transporter DcuB, which exchange fumarate against succinate, has a size of 1341 bp. We amplified the genome area of the <i>dcuB</i> gene with primers (<a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon1">dcuB_del_kon1</a> and <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon2">dcuB_del_kon2</a>), which bind 530 bp upstream and 520 bp downstream of the <i>dcuB</i> gene. When using the <i>E. coli</i> KRX wildtype as a template the resulting PCR product has a size of 2391 bp, which could be demonstrated by agarose gelelectrophoresis.
| + | |
| − | However the <i>E. coli</i> KRX knockout strain (<a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a>) showed a 4046 bp PCR product analyzed by agarose gelelectrophoresis. Figure 1 shows the result of the agarose gelelectrophoresis with <i>E. coli</i> KRX wildtype and the modified strain <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a>.<br>
| + | |
| − | <br>
| + | |
| − | The PCR product of <i>E.coli</i> KRX ΔdcuB::oprF genome amplified with primer <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon1">dcuB_del_kon1</a> and <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dcuB_del_kon2">dcuB_del_kon2</a> (4046 bp) and is composed of the antibiotic cassette (1637 bp), <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a> (1359 bp) and upstream and downstream spacer elements (520 bp and 530 bp).
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | DNA sequencing of deletion cassette from <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> showed the expected results.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | | + | |
| − | <h4 id="NPNResult">NPN-Assay</h4>
| + | |
| − | <p>
| + | |
| − | The funtionality of the outer membrane porin OprF (<a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>) in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> was investigated with a <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NPNAssay">NPN-Uptake-Assay</a> (<a href="#Cheng2005">Cheng <i>et al.</i>, 2005</a>).
| + | |
| − | <br>
| + | |
| − | 1-N-Phenylnaphthylamine (NPN) changes fluorescence activity between aqueous and hydrophobic milieus. There is only minor fluorescence in aqueous solution, but the transport in the hydrophobic periplasmatic space causes an increased fluorescence. So NPN fluorescence is a good indicator for membrane permeability. Expression of the outer membrane porines OprF effect an increasing membrane permeability.(<a href="#Loh1984">Loh <i>et al.</i>, 1984</a>)
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:600px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/6/60/Bielefeld-CeBiTec_14-10-15_NPN-Assay.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/6/60/Bielefeld-CeBiTec_14-10-15_NPN-Assay.jpg" width="600px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 2</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NPNAssay">NPN-Uptake-Assay</a>. Comparison between <i>Escherichia coli</i> KRX wildtype and <i>Escherichia coli</i> KRX with <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>, <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172502">BBa_K1172502</a> and <i>Escherichia coli</i> KRX with genome integrated <i>oprF</i> gene (<a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a>).</font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | Figure 2 shows the results of the NPN-Uptake-Assay. Higher fluorescence emission and higher membrane permeability could be observed with increasing promotor strength for <i>oprF</i>. The genome integrated <i>oprF</i> shows mid-level fluorescence emission and membrane permeability. Highest fluorescence levels could be measured with the <i>oprF</i> gene on a high-copy <a href="https://parts.igem.org/Part:pSB1C3">pSB1C3 plasmid</a> under control of the T7 promotor. <br>
| + | |
| − | Genome integrated <i>oprF</i> showed almost the same membrane permeability as the constitutive expressed <i>oprF</i> with <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K1172507">BBa_K1172507</a>. There even seems to be a marginal higher expression of the genome integrated <i>oprF</i> in contrast to the plasmid coded <i>oprF</i> although the same constitutive promotor (<a href="https://parts.igem.org/Part:BBa_J23104">BBa_J23104</a>) is used. This could be explained by physiological condition of the cell. Constitutive expression of the high-copy <i>oprF</i> causes cell stress. Protein expression could be automatically downregulated by the cells. The single-copy genome integrated <i>oprF</i> showed a reduced expression level, therefor cell stress is also smaller. As a consequence <i>E. coli</i> cells showed a more effective expression of the outer membrane porin OprF adjusted on physiological cell condition.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h4>Phenotypic characterization with Biolog® system</h4>
| + | |
| − | <p>
| + | |
| − | <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog® Microbial ID System</a> is a simple and fast method for the characterization of different bacteria, yeast and fungi species. It is based on respiration in the presence of different metabolic relevant substances for example carbon sources, nitrogen sources or toxines. The system uses redox chemistry wherein a tetrazolium dye is reduced when significant respiration occurs. Tetrazolium dye change its colour, because the cells generate reductive conditions in the course of the electron transport chain. A tetrazolium cation is reduced to formazan by dehydrogenase from the respiratory complex I. This allow a statement if respiration occurs in the presence of different substances.
| + | |
| − | <br><br>
| + | |
| − | | + | |
| − | The <i>E. coli</i> KRX wildtype and <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> were incubated in the Biolog® system to test respiration in the presence of fumarate. It was expected that the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> shows no activity in the presence of fumarate because of the knockout of the C4 carboxylate transporter <i>dcuB</i>, which makes fumarate uptake impossible for <i>E. coli</i> cells. <br>
| + | |
| − | The results of the Biolog® analysis are shown in figure 3.
| + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:800px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/f/f7/Bielefeld-CeBiTec_14-10-16_Biolog_Auswertung_Antiporter.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/f7/Bielefeld-CeBiTec_14-10-16_Biolog_Auswertung_Antiporter.jpg" width="800px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 3</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog® analysis</a> of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> in comparison to <i>Escherichia coli</i> KRX wildtype. Respiratory activity is shown under influence of fumarate.</font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | <br>
| + | |
| − | | + | |
| − | <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BiologSystem">Biolog® analysis</a> showed that there is no significant respiratory activity of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a> in the presence of fumarate during the first 35 hours of incubation. Later on respiration occurs up to 50% of maximal respiratory activity of the <i>E. coli</i> KRX wildtype after 48 hours of incubation at 37°C. <br>
| + | |
| − | The results in figure 3 demonstrate that the knockout of C4 carboxylate antiporter <i>dcuB</i> was successful. Respiration activity after 35 hours could be explained by a change in metabolism of <i>E. coli</i> cells. Based on cell stress in minimal medium bacteria use other ways for uptake of fumarate. It is possible that another transporter compensates the activity of the DcuB transporter, but activation and expression of alternative pathways require a space of time, espacially under limited conditions in minimal medium. This could explain the delayed respiration of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E.coli</i> KRX ΔdcuB::oprF</a>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | | + | |
| − | <h4>Anaerobic cultivation</h4>
| + | |
| − | <p>
| + | |
| − | We cultivated <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> under <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic conditions</a>. We used <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium">M9 minimal medium</a> with 50 mM glucose. Among growth characteristic we focused on metabolite analysis. Under anaerobic conditions <i>E. coli</i> cells use different alternative electron acceptors instead of oxygen. In part the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential pathways for bacteria to release their electrons, for example anorganic compounds like nitrate (NO<sub>3</sub><sup>-</sup>) or sulfate (SO<sub>4</sub><sup>2-</sup>).(<a href="#Gottschalk1986">Gottschalk <i>et al.</i>, 1986</a>) We analyzed succinate concentration and glucose concentration in the culture supernatant via HPLC.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | We expect succinate production of the <i>E. coli</i> KRX wildtype, but <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> should not release succinate into the media because the C4 carboxylate transporter DcuB is knocked out. As a positive control we use <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#deltadcuBKEIO"><i>E. coli</i> ΔdcuB749::kan</a>, an <i>E. coli</i> strain with a reviewed knockout in <i>dcuB</i> gene. This strain also should not show any succinate transport into the medium.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | | + | |
| − | | + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:800px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/1/12/Bielefeld-CeBiTec_14-10-15_WT_Glucose_anaerob.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/1/12/Bielefeld-CeBiTec_14-10-15_WT_Glucose_anaerob.jpg" width="800px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 4</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> of <i>Escherichia coli</i> KRX wildtype in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium">M9 minimal medium</a> with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.</font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:800px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/9/94/Bielefeld-CeBiTec_14-10-15_KEIO_Glucose_anaerob.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/94/Bielefeld-CeBiTec_14-10-15_KEIO_Glucose_anaerob.jpg" width="800px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 5</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#deltadcuBKEIO"><i>Escherichia coli</i> ΔdcuB749::kan</a> in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium">M9 minimal medium</a> with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.</font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:800px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/4/4c/Bielefeld-CeBiTec_14-10-15_deltadcuB-oprF_Glucose_anaerob.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4c/Bielefeld-CeBiTec_14-10-15_deltadcuB-oprF_Glucose_anaerob.jpg" width="800px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 6</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>Escherichia coli</i> KRX ΔdcuB::oprF</a> in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium">M9 minimal medium</a> with 50 mM glucose. Glucose and succinate concentration were measured in duplicates with HPLC.</font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | <br>
| + | |
| − | <center>
| + | |
| − | <div class="element" style="margin:10px; padding:10px; text-align:center; width:800px">
| + | |
| − | <a href="https://static.igem.org/mediawiki/2014/0/08/Bielefeld-CeBiTec_14-10-15_Vergleich_WT_Deletionsstamm_anaerob.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/08/Bielefeld-CeBiTec_14-10-15_Vergleich_WT_Deletionsstamm_anaerob.jpg" width="800px"></a><br>
| + | |
| − | <font size="2" style="text-align:left;"><b>Figure 7</b>: Results of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>Escherichia coli</i> KRX ΔdcuB::oprF</a> in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium">M9 minimal medium</a> with 50 mM glucose as compared to <i>Escherichia coli</i> KRX wildtype. Glucose and succinate concentration were measured in duplicates with HPLC. </font>
| + | |
| − | </div>
| + | |
| − | </center>
| + | |
| − | <br>
| + | |
| − | Figure 4 shows the production of succinate by the <i>Escherichia coli</i> KRX wildtype under anaerobic condition as expected. Succinate is released into the medium via the C4 carboxylate transporter DcuB. Our constructed <a href="http://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 displayed in figure 6. This demonstrates a successful knockout of the <i>dcuB</i> gene in the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> strain. As positive control we got an <i>E. coli</i> strain from the <a href="http://cgsc.biology.yale.edu/Strain.php?ID=109221">KEIO collection</a> with a verified knockout in the <i>dcuB</i> gene.<br>
| + | |
| − | Figure 5 shows the result of the <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#AnaerobicCultivation">anaerobic cultivation</a> and succinate concentration measurement of this <i>E. coli</i> strain. A very low succinate concetration in the culture supernatant could be observed. In comparison to the <i>E. coli</i> KRX wildtype there is a very low succinate export. Only traces of succinate could be measured attributed to transport of succinate by other transporters with reduced specifity. <br>
| + | |
| − | Furthermore in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#deltadcuBKEIO"><i>Escherichia coli</i> ΔdcuB749::kan</a> the knockout is not over the complete <i>dcuB</i> gene. Insertion of kanamycin selection cassette was executed at position 749 of <i>dcuB</i> gene. So there could be a residual activity of DcuB because of residual expressed parts of the antiporter protein. However the knockout of the <i>dcuB</i> in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> is complete and residual activity is not possible as shown in figure 7. Comparison between the <i>E. coli</i> wildtype and <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#KRXdeltadcuB"><i>E. coli</i> KRX ΔdcuB::oprF</a> is an obvious demonstration of a completely functional knockout of the C4 carboxylate antiporter DcuB.
| + | |
| − | | + | |
| − | </html>
| + | |
| − | | + | |
| − | ===References===
| + | |
| − | <html>
| + | |
| − | <li id="Janausch2001">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Janausch, 2001. Rekonstitution des Fumaratsensors DcuS in Liposomen und Transport von Fumarat und Succinat in Escherichia coli. <a href="http://ubm.opus.hbz-nrw.de/volltexte/2002/265/pdf/diss.pdf" target="_blank">Doctoral dissertation at Johannes Gutenberg-Universität Mainz, Germany</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | | + | |
| − | <li id="Iverson1999">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Iverson <i>et al.</i>, 1999. Structure of the <i>Escherichia coli</i> Fumarate Reductase Respiratory Complex. <a href="http://www.sciencemag.org/content/284/5422/1961.short" target="_blank">Science, vol. 284, pp. 1961-1966</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | <li id="Cheng2005">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Cheng <i>et al.</i>, 2005. New antibiotic peptides, useful in treating or preventing a microbial or viral infections or in inactivating Gram-positive and -negative bacteria, protozoa, fungi and virus. <a href="http://worldwide.espacenet.com/publicationDetails/biblio?CC=DE&NR=10360435A1&KC=A1&FT=D" target="_blank">patent DE10360435 (A1) ― 2005-07-28</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | <li id="Loh1984">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Loh <i>et al.</i>, 1984. Use of the fluorescent-probe 1-Nphenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer-membrane of Pseudomonas aeruginosa. <a href="http://aac.asm.org/content/26/4/546.short|" target="_blank">Antimicrob. Agents Chemother., vol. 26, pp. 546-551</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | <li id="Gottschalk1986">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Gottschalk <i>et al.</i>, 1986. Bacterial Metabolism. <a href="http://onlinelibrary.wiley.com/doi/10.1002/food.19870310532/abstract" target="_blank">Springer-Verlag, New York, Berlin, Heidelberg, Tokyo</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | | + | |
| − | <li id="Park1999">
| + | |
| − | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
| + | |
| − | <div id="text">
| + | |
| − | Park <i>et al.</i>, 1999. Utilization of Electrically Reduced Neutral Red byActinobacillus succinogenes: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation. <a href="http://onlinelibrary.wiley.com/doi/10.1002/food.19870310532/abstract" target="_blank">Journal of Bacteriology, vol. 181, pp. 2403-2410</a>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | | + | |
| | </html> | | </html> |
