Team:Carnegie Mellon/improvedpart
Improved Part.
Making a better estrogen sensor.
New Estrogen Sensor
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.
Improvements
Last year's sensor 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. We were unable to get any significant red fluorescent signal from our sensor cells in the presence of estrogen last year. The current version of the sensor which does not use an intein and was positioned between residues 179 and 180 of T7 RNAP and was able to give us significant fluorescent signal in the presence of estrogen. The sensor is now functional and successfully detects estrogen whereas the previous version did not.
Controls
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.
Experiments
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.
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.
Further Improvements
The reporter plasmid was then modified to express Gaussia luciferase.
References
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.