Team:METU Turkey/Results Discussion/Characterization

From 2010.igem.org

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                       <a href="http://2010.igem.org/Team:METU_Turkey/Conclusion">Conclusions</a> |
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                       <a href="http://2010.igem.org/Team:METU_Turkey/Conclusions">Conclusions</a> |
                       <a href="http://2010.igem.org/Team:METU_Turkey/Future_Work">Future Work</a> |
                       <a href="http://2010.igem.org/Team:METU_Turkey/Future_Work">Future Work</a> |
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<br> 1. Isothermal Titration Calorimetry (ITC)
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<br> <h2>1. Isothermal Titration Calorimetry (ITC)</h2>
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<br> Result: We have to cancel the ITC experiment  since we did not  handle enough DNA components ,that are promoters and response elements, and protein ,CooA .  
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<br> <h3>Result:</h3> We have to cancel the ITC experiment  since we did not  handle enough DNA components ,that are promoters and response elements, and protein ,CooA .
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<br> 2. Electrophoretic Mobility Shift Assay (EMSA)
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<br> <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/ITC"><strong>Details...</strong></a></div></html>
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<br> Result: As indicated, slight retardation in PCOOF and PCOOM bands are observed. On the other hand, it is difficult to conclude an affinity difference between these promoters based on the intensity of retarded bands.
 
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<br> 3. Intrinsic Tryptophan Fluorescence (ITF)
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<br> <h2>2. Electrophoretic Mobility Shift Assay (EMSA)</h2>
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<br> pink:reduced - CO bounded CooA + buffer  
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<br> <h3>Result:</h3> As indicated, slight retardation in PCOOF and PCOOM bands are observed. On the other hand, it is difficult to conclude an affinity difference between these promoters based on the intensity of retarded bands.
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<br> yellow : reduced –CO bounded CooA + 28 nM pCooF  ( 20. Min)
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<br> blue: reduced –CO bounded CooA +56 nM pCooF ( 40. Min)  
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<br> green: NC: reduced –CO bounded CooA + 10 ul elution buffer ( 20.min)  
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<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/EMSA"><strong>Details...</strong></a></div></html>
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<br> purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer  (40. Min )
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="http://2010.igem.org/wiki/images/b/b8/D10.jpg"></a></div></html>
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<div>
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<br> <h2>3. Intrinsic Tryptophan Fluorescence (ITF)</h2>
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="http://2010.igem.org/wiki/images/b/b5/D13.jpg"></a></div></html>
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<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/ITF"><strong>Details...</strong></a></div></html>
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<br> <h3>Result:</h3>
 +
<br> pink:reduced - CO bounded CooA + buffer
 +
<br> yellow : reduced –CO bounded CooA + 28 nM pCooF  ( 20. Min)  
 +
<br> blue: reduced –CO bounded CooA +56 nM pCooF ( 40. Min) </br>
 +
<br> green: NC: reduced –CO bounded CooA + 10 ul elution buffer ( 20.min)
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<br> purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer  (40. Min )  
<br> As shown in the graph, there is an decrease in the intensity of the fluorescence; however when looked at negative control measurements, we saw the decrease in intensity because of the dilution and time effects. So we cannot say that the reduction is caused from pCooF – CooA interaction. Moreover, we suspect that heme group in the CooA quench fluorescence of the tryptophan and also heme group gives absorbance at 280- 400 nm so our protein might give the fluorescence at that range itself.
<br> As shown in the graph, there is an decrease in the intensity of the fluorescence; however when looked at negative control measurements, we saw the decrease in intensity because of the dilution and time effects. So we cannot say that the reduction is caused from pCooF – CooA interaction. Moreover, we suspect that heme group in the CooA quench fluorescence of the tryptophan and also heme group gives absorbance at 280- 400 nm so our protein might give the fluorescence at that range itself.

Latest revision as of 03:21, 28 October 2010

Home

 


1. Isothermal Titration Calorimetry (ITC)


Result:

We have to cancel the ITC experiment since we did not handle enough DNA components ,that are promoters and response elements, and protein ,CooA .
Details...



Contents

2. Electrophoretic Mobility Shift Assay (EMSA)


Result:

As indicated, slight retardation in PCOOF and PCOOM bands are observed. On the other hand, it is difficult to conclude an affinity difference between these promoters based on the intensity of retarded bands.



Details...

w7



3. Intrinsic Tryptophan Fluorescence (ITF)

w7



Result:


pink:reduced - CO bounded CooA + buffer
yellow : reduced –CO bounded CooA + 28 nM pCooF ( 20. Min)
blue: reduced –CO bounded CooA +56 nM pCooF ( 40. Min) </br>
green: NC: reduced –CO bounded CooA + 10 ul elution buffer ( 20.min)
purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer (40. Min )


As shown in the graph, there is an decrease in the intensity of the fluorescence; however when looked at negative control measurements, we saw the decrease in intensity because of the dilution and time effects. So we cannot say that the reduction is caused from pCooF – CooA interaction. Moreover, we suspect that heme group in the CooA quench fluorescence of the tryptophan and also heme group gives absorbance at 280- 400 nm so our protein might give the fluorescence at that range itself.