RESUMEN
BACKGROUND: Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility. RESULTS: To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library. CONCLUSION: Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).
Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/análisis , Ensayos Analíticos de Alto Rendimiento , Histonas/metabolismo , Técnicas de Sonda Molecular , Sondas Moleculares/biosíntesis , Secuencia de Aminoácidos , Animales , Escherichia coli/genética , Lisina/metabolismo , Metilación , Ratones , Modelos Moleculares , Sondas Moleculares/genética , Datos de Secuencia Molecular , Biblioteca de Péptidos , Bibliotecas de Moléculas PequeñasRESUMEN
Genetically encoded biosensors allow the noninvasive imaging of specific biochemical or biorecognition processes with the preservation of subcellular spatial and temporal information. Aequorea green fluorescent protein (FP) and its engineered variants are a critical component of genetically encoded biosensors, as they serve to provide a 'read-out' of the biorecognition event under investigation. The family of FP-based biosensors includes a diverse array of designs that utilize various photophysical characteristics of the FPs. In this review, we will discuss these designs and their read-outs through reviewing some of the recent works in this area.