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Team:Carnegie Mellon/Our Sensor
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<h1> Our Sensor </h1> | <h1> Our Sensor </h1> | ||
<h2> Sensor That Reports Endocrine Activating Molecules </h2> | <h2> Sensor That Reports Endocrine Activating Molecules </h2> | ||
| - | <p> | + | <p><b> Background:</b> </p> |
| - | + | <p> Currently a method to measure estrogenic compounds with eukaryotic cells already exists, using <i>S. cerevisiae</i> with an estrogen-binding domain of the human estrogen receptor alpha. However, this yeast estrogen-screening assay (YES assay) is slow in detecting estrogen, requiring several days of incubating reporter cells to see results <sup>[1]</sup>. </p> | |
| - | + | <p>Another method is a bacterial beta-galactosidase assay to detect estrogenic compounds instead <sup>[1]</sup>. This method inserted the estrogen sensitive VMA intein into the constitutively expressed lacZ gene, and then utilized a beta-galactosidase assay to produce a signal indicating the presence of estrogenic compounds. However, this assay was not sensitive and required a substrate. </p> | |
| - | + | <p/>We also looked into a method that split T7 RNA Polymerase (T7 RNAP) with a temperature sensitive intein, creating a temperature sensitive mutant <sup>[2]</sup>. This would result in transcription of the T7 promoter and terminator only at permissive temperatures. 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, a strong viral polymerase requiring no additional factors. 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. Production of functional T7 RNAP would be reported using a fluorescent protein. </p> | |
| - | + | <p><b>References:</b></p> | |
| - | + | <p>[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.</p> | |
| - | + | <p>[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. </p> | |
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Revision as of 15:59, 30 September 2014
Project Title.
Our project description...
Our Sensor
Sensor That Reports Endocrine Activating Molecules
Background:
Currently a method to measure estrogenic compounds with eukaryotic cells already exists, using S. cerevisiae with an estrogen-binding domain of the human estrogen receptor alpha. However, this yeast estrogen-screening assay (YES assay) is slow in detecting estrogen, requiring several days of incubating reporter cells to see results [1].
Another method is a bacterial beta-galactosidase assay to detect estrogenic compounds instead [1]. This method inserted the estrogen sensitive VMA intein into the constitutively expressed lacZ gene, and then utilized a beta-galactosidase assay to produce a signal indicating the presence of estrogenic compounds. However, this assay was not sensitive and required a substrate.
We also looked into a method that split 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 permissive temperatures. 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, a strong viral polymerase requiring no additional factors. 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. Production of functional T7 RNAP would be reported using a 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.
