Team:PennState/Project

The human PPAR has three different types α, β, and γ but only two show any affect by phthalates. We are using the alpha form which is expressed in the liver, kidney, heart, muscle, adipose tissue, and others. 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 the heat shock proteins involved with synthesis in the human body. The cells that have the PPAR plasmid will be grown on plates containing Timentin which prevents growth of bacteria without plasmid. The expression of the PPAR and RXR also need tight requlation so the arabinose operon will be used. A green fluorescent protein will be placed after the PPRE to signal transcription after heterodimer binding.

The Nuclear Fusion project currently has two similar directions that it may turn but both involve a plasmid construct very generously donated to our iGEM team from David W. Wood, Department of Chemical Engineering at Princeton University. Research in their lab has constructed a biosensor containing just the ligand binding domain (LBD) of the estrogen receptor (ER). The ER is very similar to the PPAR and other hormone receptors. Previous attempts at isolating the LBD failed due to the specific folding pattern of this region required to maintain similar binding characteristics to natural ER. The folding pattern was kept by inserting the LBD into a minimal splicing intein domain. This construct was also made more soluble by addition of a maltose-binding tag. The ER was fused with a thymidylate synthase enzyme (TS) that remains deactivated until homodimerization of the ER after binding ligand. The cells are grown on thymine-free plates allowing for recognition of strength and function of ER ligands.

Our plan for this project is to work on the sensitivity of the biosensor in hopes of using this for water prescreens, similar to the Smart Fold Reporter project. The sensitivity will be focused for BPA which has a very different conformation than the natural agonist of the ER system. This difference causes BPA to bind weakly but still disturbs normal ER function. One idea is to replace the ER LBD from Wood’s biosensor with the estrogen-related receptor (ERR) LBD. The ERR is similar to ER and binds many of the same ligands and has a tendency to bind to the estrogen response element (ERE) in the human body. The one benefit of ERR for our project is that it binds BPA very strongly. Another direction that this project could take would be to analyze the LBD of ER and perform directed evolution to increase BPA sensitivity. During directed evolution, certain regions of the ER LBD would be targeted for random mutagenesis providing a library of mutants in the trillions. The mutant library would be induced with BPA and the best growing colony would be selected, tested, and mutated for further sensitivity.

Cellulosic biomass is an abundant and inexpensive energy source, coming from plant waste: ideal for Ethanol production through fermentation. However, biomass contains glucose and xylose sugars in relatively equal ratios, preferentially e. coli metabolizes glucose before any other sugar. In this project we attempt to eliminate this phenomenon, called diauxie, and get our cells to utilize both sugars at the same time. Solving this problem will lead to more efficent use of cellulosic biomass including moving towards the future of bioproduction continous processes.