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Team:NTNU Trondheim
From 2011.igem.org
Introduction
The Norwegian University of Science and Technology (NTNU) is the first Norwegian university to enter the iGEM competition. We are happy to be able to compete in this cool event, and are really excited to learn more about synthetic biology. Our project idea revolves around using the alarmone ppGpp to detect stress in E. coli, and basically make it turn red with anger (as seen in cartoons).
Project description
The idea
The stringent response in bacteria is caused by amino-acid starvation, fatty acid limitation, iron limitation, heat shock and other stress conditions. As a response under these conditions, in vitro studies have suggested that the alarmone guanosine tetraphosphate (ppGpp) increase to modulate transcription to promote survival. The increase in ppGpp levels causes a redirection of transcription so that genes important for survival are favoured at the expense of those required for growth and proliferation [1].
So, could we use ppGpp as signal molecule to find out when cells are stressed?
The solution
Our system will be based on a promoter that is important for regulating growth and proliferation. At the moment we are trying to use the rrnB-p1 promoter, which has been shown in earlier studies to be highly regulated by the ppGpp molecule. Hopefully the promoter will be down regulated enough by increased levels of ppGpp to turn the repressor lacI it controls completely off. The lacI represses a second promoter lacP that induces the production of a red fluorescent protein (mCherry) and turns the cells red. For a detailed overview of our system, see the figure below.
Optimal conditions: Under optimal conditions the rrnB P1 promoter promotes transcription of green fluorescent protein (GFP) and lacI. LacI represses the lacI/pL promoter and leads to no further transcription. Green germs.
Stressful conditions: Under stressful conditions the ppGpp concentration of the cells will increase and lead to repression of the rrnB P1 promoter. The lacI/pL promoter will no longer be repressed and start the transcription of mCherry. Red germs.
Reference:
http://www.sciencedirect.com/science/article/pii/S0966842X05000788 1 Magnusson, L. U., A. Farewell, et al. (2005). "ppGpp: a global regulator in Escherichia coli." Trends Microbiol 13(5): 236-242
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