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Wiki/smartfold/overview
From 2008.igem.org
Smart Fold, In Depth
The human <acronym title="Peroxisome Proliferator Activated Receptor">PPAR</acronym> has three different types α, β, and γ but only two are activated by phthalates. We are using the alpha form which is expressed in the liver, kidney, heart, muscle, adipose tissue, and other areas. There are different regions associated with nuclear hormone receptors: N-terminal, DNA binding domain (DBD), Hinge, Ligand binding domain (LBD), and C-terminal. The LBD is the region that attracts and holds the ligand of interest. After ligand binding the receptor usually will form a dimer, in our case PPAR will combine with Retinoid X Receptor (RXR) to form a heterodimer. The RXR protein functions much like the PPAR but in this case it does not need to attach a ligand before dimerization. The heterodimer will bind to Peroxisome Proliferator Response Element (PPRE) via the DBD and activates transcription. Most often a coactivator complex is required for transcriptional activation which involves proteins SRC-1, CBP and others.
This Smart Fold Reporter project uses altered growth conditions so that the entire PPAR protein is successfully expressed and used to transcriptionally report for the presence of phthalates. Expressing the entire PPAR in E. Coli has proven difficult which could be caused by toxicicity to the cells from the DBD. To overcome this problem we are going to treat the E. Coli with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) which is an uncoupler of oxidative phosphorylation. This strategy would correlate to a heat shock response involved with synthesis in the human body. The cells that have the PPAR plasmid will be grown on plates containing Timentin (which is a combination of antibiotic Ticarcillin and clavulanic acid) which prevents growth of bacteria without plasmid more strongly than Ampicillin alone. The expression of the PPAR and RXR also need tight regulation so the arabinose operon will be used. A green fluorescent protein will be placed after the PPRE to signal transcription after heterodimer binding.
