Team:METU Turkey/Results Discussion/Cell Sensor Experiments
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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. | <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>Myoglobin assay optimization |
Revision as of 17:48, 27 October 2010
2- Cell Sensor Experiments
- 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. 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; |
'''Cloning'''
- Sequencing: 5 of parts have been sequenced and 1 one the parts was completely wrong sequence. PcooM-RBS-RFP–TT part doesn’t have the right sequence. For this reason, we have cloned this part again and sent to IGEM headquarters. However, we didn’t have enough time for sequencing of other parts. - Agarose gel Electrophoresis: We have observed exact bands on agarose gel for each part. - Fluorescence Spectrophotometer and Confocal Laser Scanning Microscopy data: Experiments done with pCooM-RBS-RFP-TT-pLac-RBS-CooA showed that RFP fluorescence is very high with CO applied samples. Those results show that our newly cloned biobrick works successfully. - Agar plates: Our Agar plates with pCooM-RBS-RFP-TT-pLac-RBS-CooA showed that because of low level of constitutive binding of CooA to pCooM promoter, we have observed low level of RFP fluorescence on agar plates. ''' Assembly of final construct''' - Construction: We have designed two different vectors, one containing pTriEx vector containing CooA and one containing our final construct. Our final construct shows to be ineffective when there is pTriEx expression vector. Owing to this, we have designed a new vector which has pLacI-RBS-CooA. However, we didn’t have enough time for complete construction of the final vector. . |