Team:Manchester-Graz/Project/Vectordesign
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Vector design
![System_new](https://static.igem.org/mediawiki/2015/d/d3/System_new_website.png)
Figure 1 Schematic representation of our quorum sensing based regulatory system.
The second QS-System we are using, CepR/I, belongs to the opportunistic pathogen Burkholderia cenocepacia. Similar to the LuxR/I system, CepR acts as an activator of its corresponding promoter, PaidA, when a certain level of octyl-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.
Our vector is designed in a way that EsaR, EsaI and CepR are constitutively expressed by the PesaS-promoter. As long as the 3OC6-HSL concentration is low enough, EsaR will additionally increase its own transcription, creating a positive feedback loop.
![HSL](https://static.igem.org/mediawiki/2015/7/7a/Manchester-Graz_HSL_website.png)
Figure 2 Homoserinelactone synthesis by EsaI and CepI.
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 PaidA – and PesaRC –promoter.
Additionally PaidA is placed upfront of PesaRC. All reporter genes can easily be replaced by any other genes by standard cloning techniques.
![System_description](https://static.igem.org/mediawiki/2015/2/23/Manchester-Graz_System_description_website.png)
2) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing- Dependent Gene Expression
3) Shong et al (2013) Directed Evolution of the Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity
4) Weingart et al (2005) Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters
in vitro
Contact us
iGEM Subteam GrazTU Graz, Institute of Molecular Biotechnology
Petersgasse 14
8010 Graz
iGEM Subteam Manchester
University of Manchester
Manchester Institute of Biotechnology
131 Princess Street
Manchester M17DN
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