RESUMEN
Plants, as sessile organisms, possess complex and intertwined signaling networks to react and adapt their behavior toward different internal and external stimuli. Due to this high level of complexity, the implementation of quantitative molecular tools in planta remains challenging. Synthetic biology as an ever-growing interdisciplinary field applies basic engineering principles in life sciences. A plethora of synthetic switches, circuits, and even higher order networks has been implemented in different organisms, such as bacteria and mammalian cells, and facilitates the study of signaling and metabolic pathways. However, the application of such tools in plants lags behind, and thus only a few genetically encoded biosensors and switches have been engineered toward the quantitative investigation of plant signaling. Here, we present a protocol for the quantitative analysis of auxin signaling in Arabidopsis thaliana protoplasts. We implemented genetically encoded, ratiometric, degradation-based luminescent biosensors and applied them for studying auxin perception dynamics. For this, we utilized three different Aux/IAAs as sensor modules and analyzed their degradation behavior in response to auxin. Our experimental approach requires simple hardware and experimental reagents and can thus be implemented in every plant-related or cell culture laboratory. The system allows for the analysis of auxin perception and signaling aspects on various levels and can be easily expanded to other hormones, as for example strigolactones. In addition, the modular sensor design enables the implementation of sensor modules in a straightforward and time-saving approach.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Técnicas Biosensibles , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Células Vegetales/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Biosensors operating based on electrical methods are being accelerated toward rapid and efficient detection that improve the performance of the device. Continuous study in nano- and material-sciences has led to the inflection with properties of nanomaterials that fit the trend parallel to the biosensor evolution. Advancements in technology that focuses on nano-hybrid are being used to develop biosensors with better detection strategies. In this sense, titanium dioxide (TiO2) nanomaterials have attracted extensive interest in the construction of electrical biosensors. The formation of TiO2 nano-hybrid as an electrical transducing material has revealed good results with high performance. The modification of the sensing portion with a combination (nano-hybrid form) of nanomaterials has produced excellent sensors in terms of stability, reproducibility, and enhanced sensitivity. This review highlights recent research advancements with functional TiO2 nano-hybrid materials, and their victorious story in the construction of electrical biosensors are discussed. Future research directions with commercialization of these devices and their extensive utilizations are also discussed.
Asunto(s)
Técnicas Biosensibles , Nanoestructuras , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Reproducibilidad de los Resultados , TitanioRESUMEN
Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.
Asunto(s)
Técnicas Biosensibles , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa Multiplex , Plantas Modificadas GenéticamenteRESUMEN
Strigolactones are key regulators of plant development and interaction with symbiotic fungi; however, quantitative tools for strigolactone signaling analysis are lacking. We introduce a genetically encoded hormone biosensor used to analyze strigolactone-mediated processes, including the study of the components involved in the hormone perception/signaling complex and the structural specificity and sensitivity of natural and synthetic strigolactones in Arabidopsis, providing quantitative insights into the stereoselectivity of strigolactone perception. Given the high specificity, sensitivity, dynamic range of activity, modular construction, ease of implementation, and wide applicability, the biosensor StrigoQuant will be useful in unraveling multiple levels of strigolactone metabolic and signaling networks.