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Sensor That Reports Endocrine Activating Molecules

Background:

Currently a method to measure estrogenic compounds with eukaryotic cells already exists, using S. cerevisiae strain BJ3505 with an estrogen-binding domain of the human estrogen receptor alpha ^[1]. However, this yeast estrogen-screening assay (YES assay) is slow in detecting estrogen. It usually takes several days to incubate the reporter cells with the water samples in order to accumulate enough reporter protein and produce a measurable signal, which is not really suitable for large-scale sample screening ^[1].

Another method is a bacterial beta-galactosidase assay (or DIER assay) using E. coli strain DIER to detect estrogenic compounds instead ^[1]. An intein, which is a splicing protein, sometimes called a protein intron, was used for this assay. When bound to its specific molecule, an intein will splice out and produce a functional protein. This method inserted the estrogen sensitive VMA intein into two sites (between the Gly122 and Cys123 residues and Ala328 and Cys329 residues) in the essential region of the constitutively expressed lacZ gene ^[1]. In the presence of estrogenic compounds (such as 17-β estradiol), the intein would bind those, and splice out, to produce a functional LacZ protein^[1]. A beta-galactosidase assay was utilized to produce a signal indicating the presence of estrogenic compounds ^[1]. However, this assay required a two hour incubation with 17-β estradiol for efficient splicing and was not very sensitive, unable to detect certain compounds such as benz[a]anthracene and pyrene ^[1]. This may be due to the E. coli cell wall and transport system selectively decreasing a particular chemical’s potency or remaining fully impermeable to it ^[1]. This assay also required a substrate, such as ONPG, to produce a color change indicating the presence of estrogen.

A third detection system in use splits the T7 RNA Polymerase (T7 RNAP) with a temperature sensitive intein, creating a temperature sensitive mutant ^[2]. This would result in transcription of the T7 promoter and terminator only at the permissive temperature of 18 °C, but not at the restrictive temperature of 37 °C ^[2]. The S. cerevisiae VMA intein was inserted in between the Ala491 and Cys492 residues of the T7 RNA Polymerase ^[2]. The target of this assay was the lacZ gene, which would be transcribed and result in blue colonies upon the production of functional T7 RNA polymerase ^[2]. A beta-galactosidase assay, using an ONPG substrate, was also required to determine the splicing of the intein and resulting transcription of the lacZ gene.

In order to construct a sensitive assay, a system to amplify the estrogen signal was required. We designed a system that inserted an estrogen sensitive intein inside T7 RNAP at the 179 and 491 residues. T7 RNAP is a strong viral polymerase requiring no additional factors, making its expression straightforward. In the presence of estrogen, functional T7 RNAP would be produced, and readily bind to the T7 promoter, resulting in signal amplification in the presence of estrogen. Splicing of the estrogen-responsive intein in the presence of estrogen would be reported using a yellow fluorescent protein and production of functional T7 RNAP would be reported using a red fluorescent protein.

References:

[1] Liang R, Zhou J, Liu J; “Construction of Bacterial Assay for Estrogen Detection Based on Estrogen-Sensitive Intein,” Applied and Environmental Microbiology, Vol. 77, No. 7; September 30, 2010.

[2] Liang R, Liu X, Liu J, Ren Q, Liang P, Lin Z, Xie X; “A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli,” Journal of Microbiological Methods 68 (2007) 497–506.