Difference between revisions of "Team:Manchester-Graz/Project/Vectordesign"
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<p> <div id="pictureright"><img src="https://static.igem.org/mediawiki/2015/d/d3/System_new_website.png" alt="System_new" width="500"><br><b>Figure 1</b> Schematic representation of our quorum sensing based regulatory system.</div></p> | <p> <div id="pictureright"><img src="https://static.igem.org/mediawiki/2015/d/d3/System_new_website.png" alt="System_new" width="500"><br><b>Figure 1</b> Schematic representation of our quorum sensing based regulatory system.</div></p> | ||
− | <p>Our vector is based on two quorum sensing (QS) systems. The EsaR/I system belongs to the plant pathogen <i>Pantoea stewartii</i>, formerly known as <i>Erwinia stewartii</i>, the causative agent of Stewart’s Wilt(1). Contrary to common QS-systems EsaR/I uses a repressor based rather than an activator-based system. EsaR binds to its corresponding binding sites on the P<sub>esaRC</sub> promoter and represses the expression of the genes under the promoter’s control (2,3). When a certain concentration of 3-oxohexanoyl-homoserinelactone (3OC6-HSL) that is produced by the EsaI-synthase, is reached, it leads to an allosteric | + | <p>Our vector is based on two quorum sensing (QS) systems. The EsaR/I system belongs to the plant pathogen <i>Pantoea stewartii</i>, formerly known as <i>Erwinia stewartii</i>, the causative agent of Stewart’s Wilt(1). Contrary to common QS-systems EsaR/I uses a repressor based rather than an activator-based system. EsaR binds to its corresponding binding sites on the P<sub>esaRC</sub> promoter and represses the expression of the genes under the promoter’s control (2,3). When a certain concentration of 3-oxohexanoyl-homoserinelactone (3OC6-HSL) that is produced by the EsaI-synthase, is reached, it leads to an allosteric conformation change in EsaR’s structure that inhibits its repressor function. We use an engineered variant of EsaR (D91G) that showed higher sensitivity towards 3OC6-HSL (3). When positioned in the -60 region of the P<sub>esaS</sub>-promoter EsaR can work as an activator too, by facilitating RNA-polymerase recruitment (2). <br><br></p> |
<p>The second QS-System we are using, CepR/I, belongs to the opportunistic pathogen <i>Burkholderia cenocepacia</i>. Similar to the LuxR/I system, CepR acts as an activator of its corresponding promoter, P<sub>aidA</sub>, when a certain level of octanoyl-homoserinelactone (C8-HSL) is reached (4). C8-HSL is produced by CepI. CepR also binds 3OC6-HSL, however will not work as an activator, as the additional two carbon-atoms are mandatory, for CepR’s RNA-Polymerase-recruiting ability (4). This way CepR works as an competitive binding site for 3OC6-HSL, that putatively allows us to reach higher cell densities and thus higher 3OC6-HSL concentration before the EsaR/I expression system gets activated. <br><br></p> | <p>The second QS-System we are using, CepR/I, belongs to the opportunistic pathogen <i>Burkholderia cenocepacia</i>. Similar to the LuxR/I system, CepR acts as an activator of its corresponding promoter, P<sub>aidA</sub>, when a certain level of octanoyl-homoserinelactone (C8-HSL) is reached (4). C8-HSL is produced by CepI. CepR also binds 3OC6-HSL, however will not work as an activator, as the additional two carbon-atoms are mandatory, for CepR’s RNA-Polymerase-recruiting ability (4). This way CepR works as an competitive binding site for 3OC6-HSL, that putatively allows us to reach higher cell densities and thus higher 3OC6-HSL concentration before the EsaR/I expression system gets activated. <br><br></p> |
Revision as of 11:48, 7 September 2015
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