Team:METU Turkey/Results Discussion/CooA Expression and Purification

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3-CooA Expression and Purification
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<br>Fermentor studies
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<br>- Culturing: These studies showed that our E.coli BL21 DE3 strain grow well until absorbance at 600 nm reaches 2.2. However, because of high growing capacity of fermentor, sufficient time is not present for Carbon monoxide induction. Therefore, we changed fermentor to flask in order to have higher induction time period of carbon monoxide
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<br>- Viability: Cells having absorbance at 0.05 have reached the stationary phase in 3 hours and starting from 4th hour, our bacteria cultures started to die.
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<br>- Reproducibility: Our data acquired from fermentor studies are not reproducible because CO was induced to death cells.
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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/2010/3/3b/Growth_curve_alpha_1.JPG"></a></div>
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<br>- Myoglobin Assay: Our data acquired from myoglobin assay is not clear because absorbance acquired from cell density have interefered with myoglogin assay data.
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<br>- CFU(Colony Forming Unit): We couldn’t observe growth of bacteria starting from 4th  hour of fermentation.
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<br>- Fluorescence measurements: Fluorescence was detected due to auto-fluorescence of CooA proteins. Since our bacteria cultures have expression vector, CooA is highly expressed which in turn causes fluorescence.</br>
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<br>Flask experiments</br>
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<br>- Culturing: We didn’t have enough time to test our final vector. Instead, we have used BBa_K352017 to test the RFP production. 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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<br>Myoglobin assay optimization</br>
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<br>- We have planned to measure CO presence with myoglobin assay. We used myoglobin purchased from Sigma to detect levels of Carbon monoxide. In our experiments, we couldn’t measure exact CO in the environment owing to the fact that sensitivity level of the myoglobin bonded with CO was not sufficient to observe absorbance at 540 nm.
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Signal quantification with fluorescence spectroscopy
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<br>Signal Quantification of pCooF-RBS-GFP-TT: </br>
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<br>For this experiment, the bacteria containing the vector having the construct and expression vector pTriEx is incubated in E.coli BL21 strain at different conditions. The results are as the following;
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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/2010/1/1b/PCooF_experiment_alpha_1_florasan_grafik.JPG"></a></div>
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<br>- From the data acquired from spectrophotometer, we observed that neither CO presence nor the presence of pCooF-RBS-GFP-TT affected GFP intensity. We acknowledged that fluorescence increase in the intensity of 510 nm was result of fluorescent nature of CooA. It is suspected that the result is dependent on our strain, BL21, caused the unexpected results. 
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Signal Quantification of pCooM-RBS-RFP-TT-pLac-RBS-CooA: We have tried another construct pCooM-RBS-RFP-TT-pLac-RBS-CooA at TOP10 strain. The results were successful. We have put our construct in different environments (flasks with different conditions) and compared the RFP fluorescence.
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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/2010/1/1b/PCooF_experiment_alpha_1_florasan_grafik.JPG"></a></div>
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</br><br>
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Imaging with confocal laser scanning microscopy</br>
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<br> Signal Visualization of pCooM-RBS-RFP-TT-pLac-RBS-CooA:
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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/2010/2/26/Confocal_sa%C4%9F.JPG "></a></div>
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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/a/ae/Confocal_sol.JPG "></a></div>
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Revision as of 17:59, 27 October 2010

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3-CooA Expression and Purification



Fermentor studies
- Culturing: These studies showed that our E.coli BL21 DE3 strain grow well until absorbance at 600 nm reaches 2.2. However, because of high growing capacity of fermentor, sufficient time is not present for Carbon monoxide induction. Therefore, we changed fermentor to flask in order to have higher induction time period of carbon monoxide
- Viability: Cells having absorbance at 0.05 have reached the stationary phase in 3 hours and starting from 4th hour, our bacteria cultures started to die.
- Reproducibility: Our data acquired from fermentor studies are not reproducible because CO was induced to death cells.

w7

- Myoglobin Assay: Our data acquired from myoglobin assay is not clear because absorbance acquired from cell density have interefered with myoglogin assay data.
- CFU(Colony Forming Unit): We couldn’t observe growth of bacteria starting from 4th hour of fermentation.
- Fluorescence measurements: Fluorescence was detected due to auto-fluorescence of CooA proteins. Since our bacteria cultures have expression vector, CooA is highly expressed which in turn causes fluorescence.

Flask experiments

- Culturing: We didn’t have enough time to test our final vector. Instead, we have used BBa_K352017 to test the RFP production. 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.
w7

Myoglobin assay optimization

- We have planned to measure CO presence with myoglobin assay. We used myoglobin purchased from Sigma to detect levels of Carbon monoxide. In our experiments, we couldn’t measure exact CO in the environment owing to the fact that sensitivity level of the myoglobin bonded with CO was not sufficient to observe absorbance at 540 nm. Signal quantification with fluorescence spectroscopy
Signal Quantification of pCooF-RBS-GFP-TT:

For this experiment, the bacteria containing the vector having the construct and expression vector pTriEx is incubated in E.coli BL21 strain at different conditions. The results are as the following;
w7

- From the data acquired from spectrophotometer, we observed that neither CO presence nor the presence of pCooF-RBS-GFP-TT affected GFP intensity. We acknowledged that fluorescence increase in the intensity of 510 nm was result of fluorescent nature of CooA. It is suspected that the result is dependent on our strain, BL21, caused the unexpected results. Signal Quantification of pCooM-RBS-RFP-TT-pLac-RBS-CooA: We have tried another construct pCooM-RBS-RFP-TT-pLac-RBS-CooA at TOP10 strain. The results were successful. We have put our construct in different environments (flasks with different conditions) and compared the RFP fluorescence.
w7


Imaging with confocal laser scanning microscopy

Signal Visualization of pCooM-RBS-RFP-TT-pLac-RBS-CooA:
w7
w7

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