Team:LASATX/Results
Project Results
T7-GFP in pCDF, pCooF-GFP in pCDF, and T7-CooA in pCR2.1 were constructed by Gibson Assembly. E. coli BL21 (DE3) cells (New England Biolabs) were transformed with pCooF-GFP+T7-CooA, pCooF-GFP, or T7-GFP. A crude anaerobic chamber was created from a radiation waste container: Inlet and outlet adaptors were drilled into the walls of the radiation waste container, the lid sealed with vaseline, and the presence of oxygen determined with anaerobic indicator strips. Cultures were grown in LB Broth to OD600 0.6, then the cells resuspended with antibiotics and IPTG. CORM-2 (Sigma) was added at a concentration of 100 µM. After 2 hr incubation in the anaerobic chamber, or 2 hr aerobic incubation for controls, cells were resuspended in PBS and their fluorescence measured at 600 nm for 25 flashes.
Data
Vectors we used: pCR2.1-TOPO TA cloning vector (AmpR) and pCDFDuet-1 (SpecR).
Cell strain: BL21(DE3)
Parts:
- T7-CooA (pCR2.1)
- pCoof-GFP (pCDF)
- T7-GFP (pCDF)
Constructs:
- Experiment =T7-CooA, pCoof-gfp
- Negative control 1 = DE3 cells only
- Negative control 2 = pCoof-gfp (no transcription factor - shows leakage of pCoof promoter)
- Positive control = T7-gfp
Tested at 0, 10, 50, 100, 200, and 500 uM CO-RM. Grew to 0.6 OD with 20% glucose, resuspended in IPTG and induced for 2 hours under anaerobic conditions. Cells washed and resuspended in PBS.
Graphs: Fluorescence normalized by OD, subtracting background fluorescence and absorbance of PBS. Bars indicate average of samples under identical conditions. Error bars indicate standard deviation.
Data analysis
Although there is a slight increase in GFP expression from 0 to 500 uM of CO-RM, comparison with the two negative controls show that there is not a significant difference between the carbon monoxide-dependent GFP expression and GFP expression independent of the carbon monoxide detector construct.
Possible Errors:
- failing to produce significant amount of CO
- detector failing to express
- detector failing to function
- anaerobic chamber not fully flushed (oxygen remaining and affecting)
Discussion
The carbon monoxide detection construct itself did not fully function as is evident from the levels of GFP expression seen in the results. However, this does not necessarily mean that the CO detection construct failed. Possible sources of error include failure of the CORM (hereby referred to as Carbon Monoxide production molecules), the anaerobic chamber not functioning, the detector failing to express, and the detector failing to function. CORM itself cannot function properly and is cytotoxic in aerobic conditions, meaning that if our makeshift anaerobic chamber had any margin of error, it is likely that the CORM would not fully function in its production of Carbon monoxide, or would perhaps vary over time and not be a reliable source. It is even possible that any oxygen content present induced certain elements of cytotoxicity and decreased transformation efficiency. The anaerobic chamber itself was constructed from a radiation tip box, and may have had places of leakage, preventing it from being a truly anaerobic chamber. Aside from lack of complete sealing (the top was sealed with vaseline which is airtight in theory but less in practice as we opened the chamber a lot), there is a certain degree of Dissolved Oxygen (DO) content that might have interfered with our ability to produce a fully anaerobic environment.
Future Plans