Difference between revisions of "Team:Carnegie Mellon/improvedpart"
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<div class = "title">Current Estrogen Sensor</div> | <div class = "title">Current Estrogen Sensor</div> | ||
<div class = "textbody">In order to test reporters and BEAM (Biosensor Emission Analysis Machine), the team's estrogen sensor from last year <a href = “#”> link to last year's wiki </a> 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.</div> | <div class = "textbody">In order to test reporters and BEAM (Biosensor Emission Analysis Machine), the team's estrogen sensor from last year <a href = “#”> link to last year's wiki </a> 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.</div> | ||
| + | |||
| + | <div class = "title">Improvements</div> | ||
| + | <div class = "textbody">Last year's sensor used an intein which had 3 components: the N-terminus of the <i>S. cerevisiae</i> 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.</div> | ||
| + | |||
| + | <div class = "title">Controls </div> | ||
| + | <div class = "textbody">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. </div> | ||
<p><div class = "title">References</div> | <p><div class = "title">References</div> | ||
Revision as of 20:03, 18 September 2015
Improved Part.
Making a better estrogen sensor.
Current 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.
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.