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iGEM Manchester - Parts


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 threshold of 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. CFP can be replaced using the NdeI restriction site in the start codon as well as the biobrick suffix.
BBa_K1670008 Tyrosinase from Marinomonas mediterranea which converts L-Tyrosine to L-DOPA. The enzyme activity of tyrosinase is enhanced by copper supplementation via the chaperone BBa_K1670009.
BBa_K1670009 Tyrosinase chaperone of M. mediterranea responsible for copper delivery to the enzyme. Aids enzymatic conversion of L-Tyrosine to L-DOPA.
BBa_K1670006 Coding sequence for an aromatic amino acid decarboxylase which catalyses enzymatic conversion of L-Tyrosine to Tyramine as well as L-DOPA to Dopamine and L-Phenylalanine to Phenylethylamine.
BBa_K1670007 Transaminase from Chromobacterium violaceum transfers an amine group from alanine to DOPAL producing pyruvate as well as dopamine.

[1] Weingart, C., White, C., Liu, S., Chai, Y., Cho, H., Tsai, C., Wei, Y., Delay, N., Gronquist, M., Eberhard, A. and Winans, S. (2005) Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro. Molecular Microbiology. 7 (2), pp 452-467
[2] Shong, J. and Collins, C. (2013) Engineering the esaR promoter for tunable quorum sensing-dependent gene expression. American Chemical Society. 2 (10), pp. 568–575.
[3] Shong, J., Huang, Y-M., Bystroff, C. and Collins, C. (2013) Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. American Chemical Society. 8 (4), pp 789–795.