# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## 4 Discussion
Here again, our data are in agreement with the previously published results using cell extracts [3]. This biosensor is likely to be a powerful tool to precisely measure 2-OG variations in the different stages of heterocyst differentiation. However, PII being a highly conserved protein in bacteria and plants that controls the expression and activity of many enzymes such as the glutamine synthetase, the acetyl-CoA carboxylase or the nitrogenase [14; 49; 50; 51; 52; 53; 54; 55], a PII-based 2-OG biosensor is probably not the most appropriate construct to measure 2-OG fluctuations in the majority of living organisms as its overproduction could impact regulatory cascades and cellular activities, not by affecting cellular 2-OG levels but rather by titratingGInB/PII partners. By contrast, the NtcA regulator is only found within the cyanobacterial phylum. In addition, to avoid any crosstalk in other organisms, the NtcA-based biosensor we constructed lacks the DNA binding domain, and therefore lost its ability to control gene expression in _Anabaena_. This biosensor is therefore most suitable for the study of 2-OG fluctuations in the majority of organisms. These biosensors represent the first step towards the development of fluorescent sensing systems for 2-OG able to measure the difference of concentration of this universal signaling molecule in single cells. Two others 2-OG biosensors (PII-TC3 and PII-TC3-R9P) based on the PII skeleton from _Synechococcus elongatus_ PCC 7942 have been described recently [56]. Theses 2-OG biosensors have also been constructed on the basis of conformational change of this loop to measure a change in FRET. The PII proteins from _Anabaena_ and _S. elongatus_ share the same size (112 amino acids) and a high similarity of sequence. However, the structures of these biosensors (PII-TC3 and PII-TC3-R9P) and the one described in our study are not identical. Indeed, in the case of the _Synechococcus_ biosensors, the fluorophore donor is inserted in the T-loop, while it is the acceptor fluorophore in the case of _Anabaena_ biosensor. Moreover, the acceptor is fused to the C-terminus of PII via the 12-amino-acid streptavidin affinity tag and the fluorophores used are different. [PERSON] et al., fused PII to the circularly permuted mCerulean and the Venus FP, instead of mTurquoise and YFP for F-mT_PII [56]. The most striking difference between the two systems is that the fluorophore is not inserted at exactly the same position in the T-loop. In the case of _Synechococcus_ PII-based biosensors, the wild-type sequence results in a too sensitive response. Therefore, a substitution has been introduced in the sequence of PII in order to obtain a relevant biosensor. In the case of the biosensor described here, the wild type sequence of PII used turned out to be compatible with physiological concentrations measure. These structural differences between the two biosensors might explain the fact that the two systems respond to different concentrations of 2-OG. Nevertheless, the F-mT_PII and F-mT_NtcA biosensors should be tested to define the best construct for the organism under investigation. In order to further improve the ability of the biosensors to measure the dynamic of 2-OG changes in vivo over extended periods of time, several parameters should be considered. First, as for many fluorophores, repetitive fluorescence excitations yield photobleaching that delays or impedes any time-lapse measurement. We already replaced the CFP donor fluorophore by mTurquoise, which has been shown to provide a higher brightness while being less subject to photobleaching. Future improvements may include switching the YFP by a better-suited fluorophore. Second, in vivo FRET measurements need to mobilize a larger set of controls as biosensor skeletons fused to a single fluorophore, in order to modulate the data in function of fluorescence leakage and heterogeneous levels of production from one cell to another. Nevertheless, the FRET measurement can also be analyzed using alternative methods.