Team:METU Turkey/Results Discussion/Cell Sensor Experiments

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2- Cell Sensor Experiments


Design and ordering of DNA parts
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.
- 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'''
- Preparation of competent cell: 2 different methods and 1 kit were used for competent cell preparation. Fermentas Transformation kit wasn’t successful at all. Although two different TOP10 Glycerol stocks were used for this procedure, the preparation of competent cells couldn’t be accomplished. After working with Fermentas kit, TSS and Rubidium Chloride methods have been tried for competent cell preparation. Rubidium chloride method was chosen for competent cell preparation

- Visualizing Small DNA fragments on agarose gel: For this purpose, two different methods have been utilized. Firstly, ethanol precipitation method was used to concentrate DNA samples prior to assembly of biobricks. Although this method was successful, owing to the fact that it has time constraints, Vacuum drying method was used for this purpose. By this procedure, we have successfully concentrated our DNA samples.

- Restriction Digestion: Two different enzyme types of Fermentas have been used for this purpose (Fast Digest-Conventional).Fast Digest enzymes have proven to be more useful because of time saving properties.

- Ligation: Ligation procedure was performed according to molar ratio of fragments.
''' Validation'''

w7
w7

- 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.
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