Monday, July 9, 2012

iGEM2011: GFP-based readout for ppGpp concentration in vivo

Measuring ppGpp concentration in individual living cells with good temporal resolution would be great. I've been musing about a possibility of doing that using RNA aptamers, but that's just musing. It seems like am iGEM team from the University of Trondheim tried setting up a GFP-based reported system, and this system is, maybe, possibly, probably, working. Somewhat

Unfortunately there is no publication. However, there is a popular article about the whole affair, in Norwegian and there is a short report on the iGEM webpage

Apropos to the technical issues that are listed in the original report, such as dramatic leakage of the GFP reporter in the absence of stress stimuli, there are several conceptual concerns. First, the system is based on translation of the GFP reporter during the stringent response, and during the stringent response translation is strongly inhibited. Second, GFP (they use red version of it, mCherry) has to mature in order to become bringt, and that takes some time - from minutes to hours, depending on the conditions and what sort of GFP variant it is. For mCherry maturation time is 15-40 minutes, and this is comparable with E. coli generation time. Therefore, first, one would expect a very pronounced lag before the SR is engaged and the corresponding readout and, second, all the fluctuations in the ppGpp concentrations happening on the timescale below 10s of minutes will be averaged out. Third, GFP is very stable, so this reporter system will have severe memory effects - once the cell has committed to stringency, it will produce GFP, and even though stringency is reversed, GFP will stay. Maybe it is possible to turn this into a feature, but I am not sure how. And, lastly, brightness of the GFP depends on the pH and redox potential of the environment, and these things change in the stressed cells.

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