Part:BBa_K352011:Experience
Primers for pcooM amplification
pCooM
forward: 5' ccgaattcgcggccgcttctagagACAGCATGACGAG
reverse: 5' cctactagtagcggccgctgcagCGCCACCTGGGCG
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.
The data shows that RFP fluorescence increases with CO induction.
Imaging with confocal laser scanning microscopy
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.
Applications of BBa_K352011
Enhanced Dynamic Range (EDR)
In a typical biphasic binding event in which strong and weak affinity binding interactions can take place between two molecules, saturation of the strong affinity site is followed by a second saturation event of the weak site. Based on this principle, we coupled strong and weak binding response elements of carbon monoxide sensing transcription factor CooA to two different signals, GFP and RFP respectively. In this way, we expect not only to detect the presence of carbon monoxide gas but also increase the dynamic range of our cell sensor using strong-weak promoter coupling.
One of the important questions we are hoping to answer is how many fold affinity difference between CooA and its response elements (RE) would be required to so that when we couple these REs we obtain the widest possible dynamic range for the CO detection?
How E-CO Sensor Works?
When CO is introduced into the medium, transcription from both strong and weak CooA responsive promoters will be initiated. Since affinity of CO bound transcription factor is higher for the strong promoter, GFP signal will dominate the RFP signal due to the higher transcription rate of the former. Increase in CO concentration will completely saturate strong promoter and after a point saturation of the second, weaker promoter will begin. As the concentration of the signal from weak promoter (RFP) increases, detected fluorescent signal will start to change from green to yellow.
User Reviews
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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