# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## 4 Discussion
Here, we determined the FRET ratio by measuring the intensity of fluorescence of the donor and the acceptor. The FLIM-based FRET technic (Fluorescence-Lifetime Imaging Microscopy) could also report the FRET. These measurements focus on changes in the lifetime of the donor, which prevents some drawbacks of the intensity-based FRET measurement, such as the signal cross-contamination and photobleaching. Each fluorophore has its own characteristic lifetime that is dependent on its molecular environment but independent of its cellular concentration. MTurquoise is a fluorophore that has a better performance in brightness but above all two close lifetimes of 3.7 ns and 3.8 ns that can be regarded as a single lifetime by contrast to CFP that possesses two lifetimes more distanced (2.3 ns and 3.0 ns). Switching the donor fluorophore CFP to mTurquoise circumvents the challenging problem to provide a stable and uniform production of the biosensors and allows measuring in vivo 2-OG levels at the cellular level. However, the FLIM approach could be either an alternative or an additional method of FRET measure. Finally, additional biosensors based on 2-OG responsive enzymes or regulators should also be engineered and characterized to expand the set of available tools, such as the GOGAT enzyme of _Bacillus subtilis_ or _Synechococcus_ sp. [57; 58] or NrpR transcriptional regulator of _Methanococcus maripaludis_[59; 60]. Such tools will not be only important for studying heterocyst differentiation in filamentous cyanobacteria, or the TCA cycle and 2-OG signaling in various organisms in which 2-OGplays important functions, but might provide insights on human diseases that are associated with 2-OG levels imbalance or perturbations, such as acute myeloid leukemia, low-grade glioma, secondary glioblastoma, or that could be cured thanks to 2-OG inhibition of angiogenesis properties [61; 62; 63; 64; 65]. **Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2075-1729/8/4/51/s1](http://www.mdpi.com/2075-1729/8/4/51/s1)]([http://www.mdpi.com/2075-1729/8/4/51/s1](http://www.mdpi.com/2075-1729/8/4/51/s1)), Figure S1: SDS-PAGE gels stained with Coomassieie Blue for recombinant biosensors expressed in _E. coli_ BL21 (DE3); Table S1: Primers and plasmids used in this article. **Author Contributions:** [PERSON]-[PERSON] and [PERSON] conceived and designed the experiments; [PERSON] and [PERSON] performed the experiments; [PERSON]-[PERSON], [PERSON], and [PERSON] analyzed the data; [PERSON] and [PERSON] wrote the paper; all authors read and approved the final manuscript. **Funding:** This research was funded by the \"Agence Nationale de la Recherche\" ANR program 07-PCV1-0028. **Acknowledgments:** The authors thank [PERSON] for careful reading of the manuscript; [PERSON] for technical assistance; [PERSON] for encouragement; and the anonymous referees for their constructive comments and suggestions. The Aix-Marseille Univ. and the CNRS support this study. [PERSON] is supported by a PhD fellowship from the China Scholarship council. **Conflicts of Interest:** The authors declare no conflict of interest.