Part:BBa_K1023004
pH biosensor
This is a recombinant red fluorescence protein pH biosensor, named pHRed, that was genetically engineered by Mathew Tantama et. al. 2011. It can exhibits dual excitation peak at 440 nm and 585 nm, which will allow for the estimation of pH based on ratiometric method by According to Mathew Tantama et. al. 2011, the measurements of pH are not affected by different buffer concentration such as Na+, K+, Mg2+, Cl−, Ca2+, HCO3−, H2O2, DTT, and different temperature (21°C to 37°C). In addition, pHRed biosensor has been specifically codon optimized for Asp. niger and also the illegal restriction sites were removed.
Usage and Biology
Regulation of intracellular pH is important for the metabolism of a functional cell as it is tightly regulated by the complex interactions between the consumption, production, transportation and buffering of H+ (Madshus 1988; Felle 1996). To maintain protein stability, enzyme and ion channel activity, growth and division, it is essential to retain pH within their physiological range of pH 7.5 (Bagar, Altenbach et al. 2009). In addition, it is important to maintain a proton gradient between the cytoplasm and mitochondria, which allow for the formation of ATP to sustain their metabolism. pHRed biosensor allows us to non-invasively quantify pH measurements in living cells by means of ratiometric method. The advantages of using a ratiometric method is that the estimation of pH is not affected by photobleaching, changes in focus of the microscope, variations in laser intensity, optical path length and illumination intensity.
The excitation peak at 585 nm and 440 nm are caused by the anionic chromophore in an acidic condition and the protonated neutral chromophore in an alkaline condition, respectively. The pH can be calculated by measuring the fluorescence intensity ratio between excitation peaks, F585 and F440 (F585/ F440); and compare it with a calibration curve, e.g. figure below (obtained from Mathew Tantama et. al. 2011).
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Fluorescence intensity ratio between excitation peaks between 585 and 440nm (Tantama et. al. 2011)</p>
Sequence and Features
The coding sequence starts from the first ATG till TAA and it has been specifically (codon) optimized for expression in <i>Aspergillus Niger.
References
1. Bagar, T., K. Altenbach, et al. (2009). "Live-Cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe." Eukaryot Cell 8(5): 703-712.
2. Felle, H. H. (1996). "Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs." Journal of Experimental Botany 47(7): 967-973.
3. Madshus, I. H. (1988). "Regulation of intracellular pH in eukaryotic cells." Biochem J 250(1): 1-8.
4. Tantama, M., Y. P. Hung, et al. (2011). "Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor." J Am Chem Soc 133(26): 10034-10037.
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