Difference between revisions of "Part:BBa K352001:Experience"
Line 2: Line 2:
  
 
<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/CooA_Expression_and_Purification"><strong><h3>Click, For overexpression and purification of CooA protein.</h3></strong></a></div></html>
 
<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/CooA_Expression_and_Purification"><strong><h3>Click, For overexpression and purification of CooA protein.</h3></strong></a></div></html>
 +
 +
<br> <h2> Electrophoretic Mobility Shift Assay (EMSA)</h2>
 +
 +
<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.
 +
 +
 +
<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/EMSA"><strong>Details...</strong></a></div></html>
 +
 +
<html>
 +
<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b8/D10.jpg"></a></div></html>
 +
 +
<br> <h2> Intrinsic Tryptophan Fluorescence (ITF)</h2>
 +
 +
<html>
 +
<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b5/D13.jpg"></a></div></html>
 +
 +
<br>  <html><div><a href="http://2010.igem.org/Team:METU_Turkey/Results_Discussion/ITF"><strong>Details...</strong></a></div></html>
 +
 +
<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) 
 +
<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.
 +
 +
 +
  
  

Revision as of 03:21, 28 October 2010




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.



w7


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.



Applications of BBa_K352001

User Reviews

UNIQ6a60cab558f47deb-partinfo-00000005-QINU UNIQ6a60cab558f47deb-partinfo-00000006-QINU