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Using Synthetic Biology methods to construct a functional estrogen biosensor based on the dimerization‐dependent Red Fluorescent Protein
Author(s) -
Case Emma,
Desharnais Brianna,
Kragh Caroline,
Neka Ganad,
RoeckleinCanfield Jennifer
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r5504
Subject(s) - estrogen receptor , estrogen , biosensor , synthetic biology , fluorescence , computational biology , green fluorescent protein , cloning (programming) , fluorescent protein , chemistry , estrogen receptor alpha , biology , bacteria , biochemistry , microbiology and biotechnology , computer science , genetics , gene , physics , cancer , quantum mechanics , breast cancer , programming language
The presence of estrogenic compounds (endocrine‐disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time‐consuming techniques such as liquid chromatography‐mass spectrometry and high performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses rational engineering to express dimerization dependent Red Fluorescent Proteins (ddRFP) fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric weakly fluorescent RFP proteins derived from mutant dTomato (Campbell et al, 2021) are strongly fluorescent upon dimerization. These RFP constructs are brought together in the cell when ERalpha dimerizes in the presence of estrogen. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for detection of estrogenic compounds in contaminated water.