Part:BBa_K3346011
ERE Based Estrogen Biosensor Circuit
Background and usage The estrogen receptors (ER𝛂 and ERꞵ), proteins from the steroid/nuclear receptor superfamily, are ligand-activated transcription enhancers. When estrogen binds to ER𝛂 or ERꞵ in the cytoplasm of the cell, the ligand-bound receptors translocate to the nucleus where they bind to segments of DNA called estrogen response elements (EREs). Once bound to the EREs, the estrogen receptors interact with the RNA polymerase transcription initiation complex to enhance the transcription of targeted genes [1]. This regulatory system has been used to create estrogen biosensors in Saccharomyces cerevisiae through the expression of human ERs and firefly luciferase controlled by EREs [2]. In this system, the level of estrogen present affects the amount of bioluminesce in a dose-dependent manner [2]. While this circuit was created in S. cerevisiae, previous work has successfully expressed human ER𝛂 in Escherichia coli (E. coli) by using an arabinose expression system, Timentin, and carbonyl cyanide m-chlorophenyl-hydrazone, an uncoupler of oxidative phosphorylation, which is used to negate the toxic effects of this expression system in E. coli [3].
This BioBrick incorporates features and components from several different previous circuits in a novel way to generate an estrogen biosensor in E. coli using an ERE sequence activation of a T7 promoter to generate green fluorescent protein (GFP). Design and use of this circuit was informed by our mathematical model. The model suggested a weak T7 promoter, so the K613003 promoter and RBS was used to generate the proper efficiency. Additionally, the model informed us that only one ERE should be used in our model, given that literature has suggested there is a synergistic effect between the number of EREs and promoter activation, with the peak at three ERE regions [1]. Finally, it is suggested that this BioBrick be used in a high copy number plasmid, ideally with a copy number of 100. The sequence for the ERE comes from Klinge et al. 2001 [1], and the sequence for GFP comes from BBa_K12550.
Endometriosis is an estrogen-dependent disease, meaning individuals with endometriosis often have significantly increased levels of estrogen in ovarian, adipose, and skin fibroblast cells [4]. As such, we decided to create our own synthetic gene circuit in E.coli that is fine-tuned to produce a signal at the concentrations of estrogen that have been clinically implicated in endometriosis. The benefit of this estrogen-responsive system is that it can be used to suggest the dosage of medications used for menstrual suppression and hormone regulation to treat the associated symptoms of endometriosis. This has the potential to be a synthetic biology approach to the non-invasive diagnosis of estrogen-dependent diseases.
References:
Klinge, C. M. (2001). Estrogen receptor interaction with estrogen response elements. Nucleic acids research, 29(14), 2905-2919.
Leskinen, P., Michelini, E., Picard, D., Karp, M., & Virta, M. (2005). Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices. Chemosphere, 61(2), 259-266.
Zhang, C. C., Glenn, K. A., Kuntz, M. A., & Shapiro, D. J. (2000). High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP. The Journal of Steroid Biochemistry and Molecular Biology, 74(4), 169-178.
Kitawaki, J., Kado, N., Ishihara, H., Koshiba, H., Kitaoka, Y., & Honjo, H. (2002). Endometriosis: the pathophysiology as an estrogen-dependent disease. The Journal of steroid biochemistry and molecular biology, 83(1-5), 149-155.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 66
Illegal BsaI.rc site found at 764
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