Visualizing the pH in Escherichia coli Colonies via the Sensor Protein mCherryEA Allows High-Throughput Screening of Mutant Libraries
Fabian Stefan Franz Hartmann, Tamara Weiß, Jing Shen, Dóra Smahajcsik, Simonas Savickas, Gerd M. Seibold
Abstract
Phenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription factor-based biosensors have emerged as the sensor type of choice. Here, the target stimulus initiates the activation of a response gene (e.g., a fluorescent protein), followed by transcription, translation, and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real time. To capture rapid intracellular processes adequately, fluorescent reporter proteins are extensively applied. However, these sensor types have not previously been used for phenotypic screenings. To take advantage of their properties, we established here an imaging method that allows application of a rapid ratiometric sensor protein for assessing the internal pH of colonies in a high-throughput manner.