Team:PennState/fusion/overview

Hormone Biosensors

Introduction

Smart Fold

Overview

Parts

References

Nuclear Fusion

Overview

Parts

References

Diauxie Elimination

Introduction

Overview

Parts

References

Nuclear Fusion In Depth

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.