"
Team:Carnegie Mellon/Our Sensor
From 2014.igem.org
| Line 374: | Line 374: | ||
<p> Currently a method to measure estrogenic compounds with eukaryotic cells already exists, <i>S. cerevisiae</i> strains with the estrogen-binding domain of the human estrogen receptor alpha bind to estrogen responsive elements and reporters are employed (Routledge and Sumpter 1996; Gaido et al. 1997; Bistan et al. 2012). 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. </p> | <p> Currently a method to measure estrogenic compounds with eukaryotic cells already exists, <i>S. cerevisiae</i> strains with the estrogen-binding domain of the human estrogen receptor alpha bind to estrogen responsive elements and reporters are employed (Routledge and Sumpter 1996; Gaido et al. 1997; Bistan et al. 2012). 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. </p> | ||
<p>Another method is a bacterial beta-galactosidase assay that uses <i> E. coli</i> strain DIER to detect estrogenic compounds (Liang et al. 2010). This strain was engineered to contain a conditionally splicing intein (see Topilina and Mills for a review of inteins). An intein is a splicing protein, sometimes called a protein intron. When bound to its specific molecule, conditional inteins will splice out and produce a peptide bond between the two parts of protein. This method inserted the ligand binding domain of the human estrogen receptor into the yeast VMA intein to form an estrogen responsive intein. This intein was tested at two sites (between the Gly122 and Cys123 residues and Ala328 and Cys329 residues) in the essential region of the constitutively expressed lacZ gene (Liang et al. 2010). In the presence of estrogenic compounds (such as 17-β estradiol), the intein would bind those, and splice out, to produce a functional <i>LacZ</i> protein. A beta-galactosidase assay was utilized to produce a signal indicating the presence of estrogenic compounds. 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. This may be due to the <i>E. coli</i> cell wall and transport system selectively decreasing a particular chemical’s potency or remaining fully impermeable to it (Liang et al. 2010). This assay also required a substrate, such as ONPG, to produce a color change indicating the presence of estrogen. </p> | <p>Another method is a bacterial beta-galactosidase assay that uses <i> E. coli</i> strain DIER to detect estrogenic compounds (Liang et al. 2010). This strain was engineered to contain a conditionally splicing intein (see Topilina and Mills for a review of inteins). An intein is a splicing protein, sometimes called a protein intron. When bound to its specific molecule, conditional inteins will splice out and produce a peptide bond between the two parts of protein. This method inserted the ligand binding domain of the human estrogen receptor into the yeast VMA intein to form an estrogen responsive intein. This intein was tested at two sites (between the Gly122 and Cys123 residues and Ala328 and Cys329 residues) in the essential region of the constitutively expressed lacZ gene (Liang et al. 2010). In the presence of estrogenic compounds (such as 17-β estradiol), the intein would bind those, and splice out, to produce a functional <i>LacZ</i> protein. A beta-galactosidase assay was utilized to produce a signal indicating the presence of estrogenic compounds. 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. This may be due to the <i>E. coli</i> cell wall and transport system selectively decreasing a particular chemical’s potency or remaining fully impermeable to it (Liang et al. 2010). This assay also required a substrate, such as ONPG, to produce a color change indicating the presence of estrogen. </p> | ||
| - | <p>In order decrease time and increase sensitivity, our approach was to use the robust T7 RNA Polymerase (T7 RNAP) and a fluorescent reporter. T7 RNAP had been used with a temperature sensitive intein (Liang et al. 2007). At the permissive temperature the intein was spliced out to form functional T7 RNAP resulting in transcription from the T7 promoter to the terminator only at the permissive temperature of 18 °C, but not at the restrictive temperature of 37 °C. The <i>S. cerevisiae</i> VMA intein was inserted in between the Ala491 and Cys492 residues of the T7 RNAP. The T7 promoter was placed upstream of the lacZ gene, and was transcribed and translated resulting in blue colonies upon the production of functional T7 RNAP.</p> | + | <p>In order decrease time and increase sensitivity, our approach was to use the robust T7 RNA Polymerase (T7 RNAP) and a fluorescent reporter. T7 RNAP had been used with a temperature sensitive intein (Liang et al. 2007). At the permissive temperature the intein was spliced out to form functional T7 RNAP resulting in transcription from the T7 promoter to the terminator only at the permissive temperature of 18 °C, but not at the restrictive temperature of 37 °C. The <i>S. cerevisiae</i> VMA intein was inserted in between the Ala491 and Cys492 residues of the T7 RNAP. The T7 promoter was placed upstream of the <i>lacZ</i> gene, and was transcribed and translated resulting in blue colonies upon the production of functional T7 RNAP.</p> |
| - | <p>In order to construct a sensitive assay, a system to amplify the estrogen signal was required. We designed a system that inserted an estrogen | + | <p>In order to construct a sensitive assay, a system to amplify the estrogen signal was required. We designed a system that inserted an estrogen responsive intein inside T7 RNAP at the 491 and 492 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. The level of the estrogen-responsive intein sensor is reported using a yellow fluorescent protein and the level of estrogen, measured by the production of functional T7 RNAP, would be reported using a red fluorescent protein.</p> |
<p><b>References:</b></p> | <p><b>References:</b></p> | ||
Revision as of 18:45, 17 October 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, S. cerevisiae strains with the estrogen-binding domain of the human estrogen receptor alpha bind to estrogen responsive elements and reporters are employed (Routledge and Sumpter 1996; Gaido et al. 1997; Bistan et al. 2012). 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.
Another method is a bacterial beta-galactosidase assay that uses E. coli strain DIER to detect estrogenic compounds (Liang et al. 2010). This strain was engineered to contain a conditionally splicing intein (see Topilina and Mills for a review of inteins). An intein is a splicing protein, sometimes called a protein intron. When bound to its specific molecule, conditional inteins will splice out and produce a peptide bond between the two parts of protein. This method inserted the ligand binding domain of the human estrogen receptor into the yeast VMA intein to form an estrogen responsive intein. This intein was tested at two sites (between the Gly122 and Cys123 residues and Ala328 and Cys329 residues) in the essential region of the constitutively expressed lacZ gene (Liang et al. 2010). 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. A beta-galactosidase assay was utilized to produce a signal indicating the presence of estrogenic compounds. 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. 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 (Liang et al. 2010). This assay also required a substrate, such as ONPG, to produce a color change indicating the presence of estrogen.
In order decrease time and increase sensitivity, our approach was to use the robust T7 RNA Polymerase (T7 RNAP) and a fluorescent reporter. T7 RNAP had been used with a temperature sensitive intein (Liang et al. 2007). At the permissive temperature the intein was spliced out to form functional T7 RNAP resulting in transcription from the T7 promoter to the terminator only at the permissive temperature of 18 °C, but not at the restrictive temperature of 37 °C. The S. cerevisiae VMA intein was inserted in between the Ala491 and Cys492 residues of the T7 RNAP. The T7 promoter was placed upstream of the lacZ gene, and was transcribed and translated resulting in blue colonies upon the production of functional T7 RNAP.
In order to construct a sensitive assay, a system to amplify the estrogen signal was required. We designed a system that inserted an estrogen responsive intein inside T7 RNAP at the 491 and 492 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. The level of the estrogen-responsive intein sensor is reported using a yellow fluorescent protein and the level of estrogen, measured by the production of functional T7 RNAP, would be reported using a red fluorescent protein.
References:
Routledge EJ, Sumpter JP. 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15, 241–248.
Gaido KW, Leonard LS, Lovell S, Gould JC, Babaï D, Portier CJ, McDonnell DP. 1997. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol. 143(1),205-12.
Bistan M, Podgorelec M, Logar RM, Tisler T. 2012. Yeast Estrogen Screen Assay as a Tool for Detecting Estrogenic Activity in Water Bodies. Food Technol. Biotechnol. 50 (4), 427-433.
Liang R, Zhou J, Liu J. 2010. Construction of Bacterial Assay for Estrogen Detection Based on Estrogen-Sensitive Intein, Applied and Environmental Microbiology; 77, 2488–2495
Topilina NI, Mills KV. 2014. Recent Advances in in vivo applications of intein-mediated protein splicing. Mobile DNA 5,5. http://www.mobilednajournal.com/content/5/1/5
Liang R, Liu X, Liu J, Ren Q, Liang P, Lin Z, Xie X. 2007. A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli, Journal of Microbiological Methods; 68, 497–506.
