Difference between revisions of "Part:BBa K352001:Experience"
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| + | <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> | ||
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| + | <br> After Purification with Q-Sepharose column of BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono. | ||
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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="https://static.igem.org/mediawiki/2010/9/93/Br14.jpg"></a></div></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="https://static.igem.org/mediawiki/2010/b/be/Br18.jpg"></a></div></html> | ||
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| + | <br> <h2> Electrophoretic Mobility Shift Assay (EMSA)</h2> | ||
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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> <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> <h2> Intrinsic Tryptophan Fluorescence (ITF)</h2> | ||
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| + | <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 500px; height: 500px;" alt="w7" src="https://static.igem.org/mediawiki/2010/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) | ||
| + | <br> purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer (40. Min ) | ||
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| + | <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. | ||
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| + | <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/e/eb/D12.jpg"></a></div> </html> | ||
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| + | <br><div align="center"> Heme group in the CooA quench fluorescence of the tryptophan due to close location</div> | ||
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| + | Reference: Andrea L, 2007, The Biophysical Characterization of the three activation states of cooA through protein unfolding studies, Phd Thesis @ WISCONSIN MEDISON UNIVERSITY | ||
===Applications of BBa_K352001=== | ===Applications of BBa_K352001=== | ||
| + | <br>We hope to provide a technical guideline for other future gas sensing cell sensor projects with our E-CO sensor with enhanced dynamic range project. | ||
===User Reviews=== | ===User Reviews=== | ||
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| + | ==Culturing CO Sensor E.coli with CO induction== | ||
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| + | <html><div align="center" style="margin:15px 0px 0px 0px"> <img style="border: 0px solid ; width: 900px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/4/49/Culturing_alpha_1.JPG"></a></div> </html> | ||
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| + | <br> We have used 300 mL flasks with 80 mL LB culture. We have applied 80 Ml Carbon monoxide to some. The results were successful. Cultures induced with carbon monoxide successfully expressed red fluorescent protein. | ||
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| + | There is a bit red color in flask without any CO exposure. This might be because cooA protein is transcriptional activator not a transcriptional factor. Therefore, a leakage have been observed in our experiments; however, there is very good differentiation which is sensible. | ||
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| + | <br> Reference: Aono S (2003). Biochemical and Biophysical Properties of the CO-Sensing Transcriptional Activator CooA. Accounts of Chemical Research 2003; 36(11): 825-831. | ||
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Latest revision as of 16:05, 28 October 2010
After Purification with Q-Sepharose column of BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.
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.
Intrinsic Tryptophan Fluorescence (ITF)
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.
Reference: Andrea L, 2007, The Biophysical Characterization of the three activation states of cooA through protein unfolding studies, Phd Thesis @ WISCONSIN MEDISON UNIVERSITY
Applications of BBa_K352001
We hope to provide a technical guideline for other future gas sensing cell sensor projects with our E-CO sensor with enhanced dynamic range project.
User Reviews
Culturing CO Sensor E.coli with CO induction
We have used 300 mL flasks with 80 mL LB culture. We have applied 80 Ml Carbon monoxide to some. The results were successful. Cultures induced with carbon monoxide successfully expressed red fluorescent protein.
There is a bit red color in flask without any CO exposure. This might be because cooA protein is transcriptional activator not a transcriptional factor. Therefore, a leakage have been observed in our experiments; however, there is very good differentiation which is sensible.
Reference: Aono S (2003). Biochemical and Biophysical Properties of the CO-Sensing Transcriptional Activator CooA. Accounts of Chemical Research 2003; 36(11): 825-831.
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