This weeks paper (Discussion taking place on 14th of August, 8 pm UK time) is a slightly different topic then what we have covered so far, more towards applied microbiology. The publication is on genetically encoded nanonsensor to monitor citrate concentrations in vivo. I chose this paper as I found the methodology presented interesting and thought it gives a nice basis for a discussion.
“Engineering Genetically Encoded Nanosensors for Real-Time In Vivo Measurements of Citrate Concentrations” (PlosOne, Vol 6, Issue 1, Dec 2011) By JC Ewald, S Reich, S Baumann, WB Frommer, N Zamboni
Background and motivation
Monitoring metabolite concentrations is an important tool in metabolomics, NMR or mass spectrometry rely on high cell density cultures and often several sample preparation steps are necessary being possible error source for a study. The publication presents the construction of a FLIP (fluorescent indicator protein) employing FRET (Foerster resonance energy transfer) to monitor citrate concentrations in vivo.
After testing several citrate binding proteins, the most promising of them CitA a sensor histidine kinase from Klebsiella pneumoniae sandwiched between CFP and Venus (a modified YFP) without any linker was characterised and optimised by removing flexible regions and introducing some amino acid substituions. By this it was possible to construct in total seven sensors with different affinities for citrate covering three orders of magnitudes without loosing specificity.
In an in vivo application the response of starved E.coli cultures to different substrates was monitored. In the experiment, it could be shown that the cells do react differently on acetate medium the citrate concentrations remain high, whereas on glucose it decreases which they explain by higher glycolytic activity on glucose than TCA cycle capacity during starvation leading to an accumulation of citrate until the downstream enzymes are induced. This shows that the sensor can elucidate different metabolic pathway uses and by using several affinities an indication of the concentration can be made.
Was the paper clearly written?
Can you think of other applications? Other useful metabolite as targets to measure?
Does this method give more/less information than standard metabolomics approaches (like MFA=metabolic flux analysis, finger/footprints)
Do you think the in vivo application experiment was worthwhile?