Difference between revisions of "Team:Manchester-Graz/Project/Vectordesign"
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<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 PaidA promoter that transcribes monomer red fluorescent protein (mRFP) as a second reporter gene. <br> | <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 PaidA 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> | ||
− | <p>We want to assemble two variants of | + | |
+ | <p>We want to assemble two variants of pCERI that only slightly differ in the positioning of the regulatory proteins EsaR and CepR on the transcript of P<sub>esaS</sub>. The first variant will be assembled according to Figure 3, while in an alternative version (v2) the genes encoding esaR and cepR are switched. Usually genes further downstream on the transcript get translated less efficiently. Thus the switch in the positioning of the regulatory proteins on the transcript might influence the expression behavior of our regulatory system </p> | ||
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Revision as of 07:57, 27 August 2015