Using Synthetic Biology methods to construct a rapid and efficient estrogen biosensor

Abstract

Synthetic Biology demonstrates the integral relationship between biology, chemistry, mathematics and computer science. The field provides innovative approaches to a wide range of applications, such as bioremediation, sustainable energy production and biomedical therapies. Synthetic biology involves construction of genetic circuits when DNA sequences are modified to include standard restriction enzyme sites making the DNA sequences interchangeable and allowing for construction of a variety of circuits. New tools and devices are constantly needed to enhance the already extensive list of BioBricks housed at the Registry of Standard Biological Parts. As proof of concept we constructed a new BioBricks based on the toluene sensor built by Stiner and Haverson (AEM, 2002) in Pseudomonas. Toluene is an environmental contaminant that is the byproduct of oil contamination. Biosensors generated from BioBrick parts yield more affordable, transportable, and faster methods of pollutant detection. The tbuT cassette from Stiner and Haverson's work was digested with XbaI and SpeI sites then ligated with the GFP reporter gene and cloned into iGEM BioBrick vectors in order to create a biosensor in Escherichia coli. These BioBricks can be readily used with a variety of reporter genes to create new versions of the biosensor. We expanded our biosensor construction to create a dual functional estrogen sensitive BioBrick. 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. Our estrogen responsive plasmid contains parts that allow for both the constitutive expression of the estrogen receptor followed by an estrogen response element toggled to a reporter gene in a single vector. When the estrogen response element is activated the reporter gene, red fluorescent protein, will produce a color change in E.coli. We will present data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for estrogenic compounds in contaminated water.Support or Funding InformationNSF grant #NSF‐GPG‐1444406, GPG: Research Collaboration: REU Pilot: Synthetic Biology Boot camp