Difference between revisions of "Team:Carnegie Mellon/improvedpart"

Steroid hormones such as estrogen can diffuse across the plasma membrane, bind their receptor in the cytoplasm, migrate to the nucleus and act as transcription factors to alter cell’s physiology and behavior. Naturally occurring steroid hormones include estrogen, progesterone, testosterone and cortisol.

Detection of hormones in the environment has raised concerns in recent years because of their potential to affect both humans and wildlife. Estrogens from natural, synthetic, plant, and fungal sources can manifest endocrine disrupting properties and even at low concentrations can have harmful effects due to receptor activation. Estrogenic activity can occur in water sources including waste, drinking and freshwater. In freshwater, estrogens are harmful to the ecosystems, feminizing fish and disrupting the overall populations of organisms in the ecosystem. Estrogenic compounds can also be present in what we drink, however since the presence of hormones in water is a relatively new area of study, there have been no previous restrictions or regulations regarding filtration of estrogenic compounds.

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.

In order to test reporters and BEAM (Biosensor Emission Analysis Machine), the team's estrogen sensor from last year was improved. The biosensor is a bacterial cell containing two-plasmids. The sensor plasmid is a high-copy plasmid, which has the ligand binding domain of the human estrogen receptor alpha (ER-LBD) inserted into T7 RNA polymerase (T7 RNAP) and YFP for normalization. When the ER-LBD binds estrogen, it causes a conformational change (McLachlan et al. 2011) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (Shis and Bennet, 2012). T7 RNAP is a strong phage RNA polymerase that requires no additional factors. The second plasmid that makes up our sensor is a low-copy plasmid, the reporter plasmid, which has the T7 promoter driving expression of RFP. When the T7 RNAP is reconstituted upon binding to estrogen, it allows for binding to the T7 promoter on the reporter plasmid and transcription of the RFP mRNA which then is translated to produce RFP.

Due to concern with the compounds in water, last year’s team developed a sensor to detect the molecules in water that will bind to the estrogen receptor. Last year's biosensor used an intein which had 3 components: the N-terminus of the S. cerevisiae VMA intein, the human estrogen receptor ligand binding domain, and the C-terminus of the intein all inserted into T7 RNAP between amino acids 491 and 492. The team was unable to get any significant red fluorescent signal from the sensor cells in the presence of estrogen last year. In order to test reporters and BEAM (Biosensor Emission Analysis Machine), the team's estrogen sensor from last year link to last year's wiki was improved. The biosensor is a bacterial cell containing two-plasmids. The sensor plasmid is a high-copy plasmid, which has the ligand binding domain of the human estrogen receptor alpha (ER-LBD) inserted into T7 RNA polymerase (T7 RNAP) and YFP for normalization. When the ER-LBD binds estrogen, it causes a conformational change (McLachlan et al. 2011) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (Shis and Bennet, 2012). T7 RNAP is a strong phage RNA polymerase that requires no additional factors. The second plasmid that makes up our sensor is a low-copy plasmid, the reporter plasmid, which has the T7 promoter driving expression of RFP. When the T7 RNAP is reconstituted upon binding to estrogen, it allows for binding to the T7 promoter on the reporter plasmid and transcription of the RFP mRNA which then is translated to produce RFP. This version of the sensor does not use an intein and was positioned between residues 179 and 180 of T7 RNAP. The improved sensor was able to provide significant fluorescent signal in the presence of estrogen. The sensor is now functional and successfully detects estrogen whereas the previous version did not.

For these experiments there were three controls that did not contain the ER-LBD. The first control was intact T7 RNAP with no YFP and the second control had YFP. The third control had restriction sites in place of the ER-LBD. The sites added the amino acids ACLKLGGSTGGGSHNC between K179 and K180.

The improved sensor and controls were tested using a variety of growth protocols to evaluate the response to estrogen. A TECAN plate reader was used to measure red and yellow fluorescence after overnight exposure to various concentrations of 17-beta-estradiol. The controls showed no response and the sensor cells showed differences in RFP signal ratioed to YFP signal at concentrations ranging from 1nM to 100 uM.

Experiments were performed to test the protocol for the estrogen sensor and the sensitivity of the sensor. The most reliable sensor protocol was using overnights from single colonies which were then restarted; this gave us consistent starting cells for the assay. There were also 3 controls that were tested as well. One control had no YFP. The second control had restriction sites in place of the estrogen receptor ligand binding domain. The third control had YFP and no restriction sites. The sensor showed a three-fold or more increase in mRFP fluorescence signal upon addition of estrogen while the controls showed relatively no increase in mRFP fluorescence signal. The fluorescence from the controls was _____ and the maximum signal from the estrogen sensor was about seven times higher than the no estrogen signal. Concentration data was also acquired. Concentrations of 100 uM, 20 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, and 0 nM beta-estradiol had their fluorescence tested. As expected, the more beta-estradiol present, the higher the mRFP signal acquired.

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.

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.

McLachlan MJ, Katzenellenbogen JA, Zhao H. 2011. A new fluorescence complementation biosensor for detection of estrogenic compounds. Biotechnol Bioeng. 108, 2794-803.

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.

Shis DL and Bennet MR. 2012. Library of synthetic transcriptional AND gatesbuilt with split T7 RNA polymerase mutants. PNAS. 110, 5028-5033.