Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level
Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level
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DOI：
10.1007/s00216-014-8165-1
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发表时间：
2014-11-01
影响因子：
4.3
通讯作者：
Karp, Matti
中科院分区：
文献类型：
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作者：
Abraham, Bobin George;Santala, Ville;Karp, Matti
监测细菌细胞的细胞内氧化还原状态可以提供有关细胞生理状态的重要信息，这些信息可用于代谢工程和计算建模等多种应用。基于荧光蛋白的基因编码传感器可用于监测细胞内氧化/还原状态。这项研究报告了使用荧光蛋白对和福斯特共振能量转移（FRET）现象进行细胞内测量的氧化还原传感器的开发。为了开发该传感器，荧光蛋白 Citrine 和 Cerulean 经过基因改造，在靠近发色团的蛋白质表面携带反应性半胱氨酸残基，并使用生物素化结构域作为连接子融合构建的 FRET 对。在氧化状态下，FRET 对通过形成不稳定的二硫键而非常接近，从而导致更高的 FRET 效率。在还原性环境中，由于 FRET 对之间的距离增加，FRET 会减弱，从而为传感器提供了较大的动态测量范围。大肠杆菌突变体的细胞内研究揭示了传感器在单细胞水平上检测实时氧化还原变化的能力。结果通过基于强度和时间分辨的测量进行了验证。还证明了基于荧光蛋白的 FRET 传感器在固体表面的功能固定用于体外应用。
Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Forster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated.