Difference between revisions of "Team:Manchester-Graz/Project/Vectordesign"
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<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> | ||
− | <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 also CepI 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>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 also CepI 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> | ||
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2) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing- Dependent Gene Expression <br> | 2) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing- Dependent Gene Expression <br> | ||
3) Shong et al (2013) Directed Evolution of the Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity <br> | 3) Shong et al (2013) Directed Evolution of the Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity <br> | ||
− | 4) Weingart et al (2005) Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters <br>in vitro | + | 4) Weingart et al (2005) Direct binding of the quorum sensing regulator CepR of <i>Burkholderia cenocepacia</i> to two target promoters <br>in vitro |
</div> | </div> |
Revision as of 08:02, 27 August 2015
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