Team:Qdai/Methodology
Methodology
Overview
Our bacteria will be engineered to detect carbon monoxide. We intend to introduce the carbon monoxide sensor protein CooA and the photoprotein luciferase into E. coli.
CooA
CooA is a carbon monoxide-dependent transcriptional activator contained in Rhodospirillum rubrum. CooA contains heme and activates CooA-dependent promoters when it binds CO.[1] CooA is active as the transcriptional activator even in E. coli cells.[2]
Firefly Luciferase
Firefly luciferase is an enzyme that catalyzes a bioluminescent reaction in the presence of Mg2+. We expect that the luciferase produced by E. coli and D-luciferin added to the culture medium will cause the following reaction.[3] (D-luciferin is the natural substrate of the enzyme luciferase.[4])
CO detection system
The animation below shows the flow of CO detection. First, when CO is present in the vicinity, the constantly produced CooA binds to the CO. Since there is a gene for luciferase downstream of pCooM, we can get the light output by adding luciferin in the medium.
Fig. 1 Animation of CO detection
Fig. 2 Our plasmid design
How to optimize sensitivity?
Since the E. coli we are engineering is intended to be used as a CO detector, we need to adjust the CO sensitivity and make it emit light at the optimal CO concentration. We believe that we can achieve this by using promoters and RBS with different strengths. For example, there are two types of CooA-dependent promoters: a strong promoter (pCooF) and a weak promoter (pCooM). By combining these with two RBS of different strengths, four different patterns can be constructed.
Fig. 3 Combination of promoter and RBS
References
[1] Aono, S., Nakajima, H., Saito, K., and Okada, M., (1996). A novel heme protein that acts as a carbon monoxide-dependent transcriptional activator in Rhodospirillum rubrum. Biochem Biophys Res Commun. 228(3):752-6.[2] Aono, S., Matsuo, T., Shimono, T., Ohkubo, K., Takasaki, H., Nakajima, H., (1997). Single transduction in the transcriptional activator CooA containing a heme-based CO sensor: isolation of a dominant positive mutant which is active as the transcriptional activator even in the absence of CO. Biochem Biophys Res Commun. 240(3):783-6.
[3] Jeffrey R. de Wet, Keith V. Wood, Marlene DeLuca, Donald R. Helinski, and Suresh Subramani, (1989) Firefly Luciferase Gene: Structure and Expression in Mammalian Cells. Molecular and Cellular Biology. 7:783-6
[4] Meroni, G., Rajabi M., and Santaniello E., (2009). D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. ARKIVOC. 2009(1): 265-88