Difference between revisions of "Part:BBa K1732018"
 
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[[File:Report Luc.jpg]]
 
[[File:Report Luc.jpg]]
  
 +
This plasmid works with BBa_K1732015 in a two plasmid system that enables the production of a gaussia luciferase output when the substrate coelenterazine is added. The reporter plasmid which was originally BBa_K1491033 was then modified to express Gaussia luciferase.
  
−
In order to test reporters and BEAM, 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 (ref) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (ref). 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.
+
----
−
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. <insert pictures of control plasmids>
+
  
−
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.
+
In order to test reporters and BEAM (Carnegie Mellon University's DIY fluorimeter and luminometer), 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 (ref) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (ref). 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 Gaussia luciferase. 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 Gaussia luciferase mRNA which then is translated to produce Gaussia luciferase.
  
 +
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.
 +
 +
 +
[[File:estrogen controls Final.jpg]]
 +
----
 +
'''Experiments with Estrogen Biosensor using Gaussia Luciferase Modified Reporter Plasmid '''
 +
 +
[[File:Michelle Chart 3.jpg]]
 +
 +
Experiments were performed to demonstrate whether the addition of beta-estradiol to the biosensor using the Gaussia luciferase modified reporter plasmid had an effect of the luminescence levels in the presence of 10 ul of 2 uM coelenterazine. Overnight cultures of the biosensor in MACH cells were grown, one with beta-estradiol (estrogen +) and one without (estrogen -). A control of J23100 RFP was used as a control because no luminescence was expected to be expressed even with the addition of coelenterazine. The biosensor in the presence of beta-estradiol showed a 5 fold increase in luciferase activity.
 +
 +
[[File:michelle chart 4.jpg]]
 +
 +
[[File:Controls Graph.jpg]]
 +
 +
[[File:estrogen graph.jpg]]
 +
 +
----
 +
'''Applications'''
 +
 +
Our improved part can now be used as a sensor to detect the presence of estrogen. Further testing could include testing the sensor with estrogenic compounds other than beta-estradiol.
 +
 +
----
 +
'''References & Sources of Sequence'''
 +
 +
1. The position within T7 RNAP was from:
 +
  Shis DL and Bennet MR. 2012. Library of synthetic transcriptional AND gatesbuilt with split T7 RNA polymerase mutants. PNAS 
 +
  110: 5028-5033.
 +
 +
2. The ER LBD was from:
 +
  McLachlan MJ, Katzenellenbogen JA, Zhao H. 2011.  A new fluorescence complementationbiosensor for detection of estrogenic
 +
  compounds. Biotechnol Bioeng 108(12):2794-803
  
−
The reporter plasmid was then modified to express Gaussia luciferase.
 
  
  

Latest revision as of 01:19, 19 September 2015

pSB3K3-T7prom-B0034-Gaussia-T7term

Estrogen1.jpg

Report Luc.jpg

This plasmid works with BBa_K1732015 in a two plasmid system that enables the production of a gaussia luciferase output when the substrate coelenterazine is added. The reporter plasmid which was originally BBa_K1491033 was then modified to express Gaussia luciferase.

In order to test reporters and BEAM (Carnegie Mellon University's DIY fluorimeter and luminometer), 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 (ref) that brings together the separated domains of T7 RNAP and the activity of the T7 RNAP is reconstituted (ref). 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 Gaussia luciferase. 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 Gaussia luciferase mRNA which then is translated to produce Gaussia luciferase.

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.


Estrogen controls Final.jpg

Experiments with Estrogen Biosensor using Gaussia Luciferase Modified Reporter Plasmid

Michelle Chart 3.jpg

Experiments were performed to demonstrate whether the addition of beta-estradiol to the biosensor using the Gaussia luciferase modified reporter plasmid had an effect of the luminescence levels in the presence of 10 ul of 2 uM coelenterazine. Overnight cultures of the biosensor in MACH cells were grown, one with beta-estradiol (estrogen +) and one without (estrogen -). A control of J23100 RFP was used as a control because no luminescence was expected to be expressed even with the addition of coelenterazine. The biosensor in the presence of beta-estradiol showed a 5 fold increase in luciferase activity.

Michelle chart 4.jpg

Controls Graph.jpg

Estrogen graph.jpg

Applications

Our improved part can now be used as a sensor to detect the presence of estrogen. Further testing could include testing the sensor with estrogenic compounds other than beta-estradiol.

References & Sources of Sequence

1. The position within T7 RNAP was from: 

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

2. The ER LBD was from: 

  McLachlan MJ, Katzenellenbogen JA, Zhao H. 2011.  A new fluorescence complementationbiosensor for detection of estrogenic 
  compounds. Biotechnol Bioeng 108(12):2794-803


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 581
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 462
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]