Team:Manchester-Graz/Basic Part
Parts
| Biobrick | Description |
|---|---|
| BBa_K1670000 | This part encodes for the homoserine lactone-synthase of the CepR/I-system from Bulkholderia cenocepacia that produces octanoyl-homoserine lactone (C8-HSL). Upstream of the start codon a ribosome-binding site is already placed. |
| BBa_K1670002 | BBa_K1670002 (CepR) is the regulatory counterpart of the CepR/I quorum sensing system of Bulkholderia cenocepacia. The protein forms inclusion bodies in the absence of C8-HSL and needs to bind C8-HSL to stay in a soluble form [1] When a concentration of 80-100 nM C8-HSL is reached, the homoserine lactone binds CepR and induces a dimerization of the regulatory protein and allows binding to its corresponding DNA-binding site in an imperfect dyad manner[1] and works as an activator of the corresponding promoter. The gene is codon optimized for E.coli BL21 and contains a synthetic ribosome binding site. |
| BBa_K1670003 | BBa_K1670003 (PaidA_mRFP) contains the CepR/I regulated aidA promoter. The CepR binding box is located 44 bp upstream of the predicted transcription start. The part also encodes for a mRFP fluorescent reporter, based on BBa_K1362461. Two silent mutations (T213A, T435A) were introduced to delete two HindIII recognition sites. mRFP can be exchanged using an XhoI restriction site directly upstream of the start codon and the biobrick suffix. |
| BBa_K1670004 | BBa_K1670004 (EsaI) encodes for the homoserine lactone – synthase of the EsaR/I system from Erwinia stewartii that produces 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). Upstream of the start codon a ribosome-binding site is already placed. |
| BBa_K1670005 | BBa_K1670005 encodes for the regulatory protein EsaR of the EsaR/I quorum sensing system from Erwinia stewartii . Contrary to most other quorum sensing systems, EsaR works as a repressor rather than an activator. It binds at its corresponding binding box between the -10 and the -35 region of its corresponding promoter and inhibits transcription. Binding of 3OC6-HSL to EsaR induced an allosteric change in the structure that prevents its DNA-binding ability and thus induces expression. If the binding box is positioned shortly upstream of the promoter, EsaR also works as an activator of the respective promoter as long as it can bind to the DNA. We use a D91G variant of the gene that shows higher sensitivity towards 3OC6-HSL [2]. |
| BBa_K1670001 | BBa_K1670001 contains the EsaR/I regulated esaRC promoter, an engineered variant of the native esaR-promoter from Erwinia stewartii that contains two EsaR-boxes at the -10 region and between the -10 and -35 region[3] that allow a tighter control of the expression of the genes under the control of PesaRC. The part already contains a codon optimized CFP based on BBa_E0020 with a synthetic ribosome-binding site as a fluorescent reporter. eCFP can be replaced using the NdeI restriction site in the start codon as well as the biobrick suffix. |
1) Weingart et al (2005) Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro; Molecular Microbiology 57 452-467
2) Shong et al (2013) Directed Evolution of Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity, ACS Chemical Biology 8 p.789-795
3) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing-Dependent Gene Expression, ACS Chemical Biology 2 p.568-575
2) Shong et al (2013) Directed Evolution of Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity, ACS Chemical Biology 8 p.789-795
3) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing-Dependent Gene Expression, ACS Chemical Biology 2 p.568-575
