Difference between revisions of "Team:Manchester-Graz/Project/Vectordesign"
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<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>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 | + | <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 a 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>Our vector is designed in a way that EsaR, EsaI and CepR are constitutively expressed by the P<sub>esaS</sub>-promoter. As long as the 3OC6-HSL concentration is low enough, EsaR will additionally increase its own transcription, creating a positive feedback loop. <div id="pictureleft" style="height:160px;"><img src="https://static.igem.org/mediawiki/2015/7/7a/Manchester-Graz_HSL_website.png" alt="HSL" width="350"><br> <b>Figure 2</b> Homoserinelactone synthesis by EsaI and CepI.</div> <p>When the 3OC6-HSL threshold is reached, transcription of the PesaRC initiates, while the P<sub>esaS</sub>-feedback loop is turned off. The activation of the promoter is shown and measured on the expression of cyan fluorescent protein (CFP). Additionally to the reporter gene | + | <p>Our vector is designed in a way that EsaR, EsaI and CepR are constitutively expressed by the P<sub>esaS</sub>-promoter. As long as the 3OC6-HSL concentration is low enough, EsaR will additionally increase its own transcription, creating a positive feedback loop. <div id="pictureleft" style="height:160px;"><img src="https://static.igem.org/mediawiki/2015/7/7a/Manchester-Graz_HSL_website.png" alt="HSL" width="350"><br> <b>Figure 2</b> Homoserinelactone synthesis by EsaI and CepI.</div> <p>When the 3OC6-HSL threshold is reached, transcription of the PesaRC initiates, while the P<sub>esaS</sub>-feedback loop is turned off. The activation of the promoter is shown and measured on the expression of cyan fluorescent protein (CFP). Additionally to the reporter gene CepI also gets expressed, resulting in the time-shifted activation of our second QS-system. When the C8-HSL threshold is reached, CepR can work as an activator of the P<sub>aidA</sub> promoter that transcribes monomer red fluorescent protein (mRFP) as a second reporter gene. <br> |
To avoid any leaky read through of transcription terminators, the constitutively expressed transcripts of the regulatory proteins of the two QS-systems as well as the beta-lactamase resistance marker, are positioned in the opposite direction of the auto-induced P<sub>aidA</sub> – and P<sub>esaRC</sub> –promoter (Figure 3). <br>Additionally P<sub>aidA</sub> is placed upfront of P<sub>esaRC</sub>. All reporter genes can easily be replaced by any other genes by standard cloning techniques. <br></p></p> | To avoid any leaky read through of transcription terminators, the constitutively expressed transcripts of the regulatory proteins of the two QS-systems as well as the beta-lactamase resistance marker, are positioned in the opposite direction of the auto-induced P<sub>aidA</sub> – and P<sub>esaRC</sub> –promoter (Figure 3). <br>Additionally P<sub>aidA</sub> is placed upfront of P<sub>esaRC</sub>. All reporter genes can easily be replaced by any other genes by standard cloning techniques. <br></p></p> | ||
Revision as of 13:02, 14 September 2015