Difference between revisions of "Part:BBa K639003"

Revision as of 15:44, 15 September 2011

rrnB P1-LacI-pLac-mCherry plausible stress sensor

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 pLac that induces the production of red fluorescent protein (mCherry) and turns the cells red.

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

Plasmid map of the complete construct. Promoters are shown in blue, and genes as inside arrows. Only single restriction sites are shown.

The promotor is a variant of BBa_K112118, that we have made ourselves using PCR, and the PCR product was directly used as insert and connected with the rest of the construct. The final construct was cut with BstBI to verify that the rrnB P1 had been insertet. The stress sensor has a total length of 2653 bp, and is located inside the plasmid pSB1A2, carrying ampicillin resistance. We have put the stress sensor through several tests (see [http://2011.igem.org/Team:NTNU_Trondheim/stress-sensor characterization])

Sequence and Features