A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors

Abstract

BackgroundFluorescent 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.ResultsTo 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.ConclusionApplication 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%).

Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals

“…The outer membrane of bacteria is easily permeable to metal ions and low molecular weight compounds, thus allowing manipulation of analyte concentration for efficient clone selection. A rapid linker optimization for split and insertion BphP variants can be achieved using a histone methylation-based system adopted for screening in E. coli colonies 76 .…”

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

“…The most optimized GECIs are single-wavelength green indicators based on the original GCaMP sensor (Nakai et al, 2001). Improvements have been facilitated both by crystal structure determination in the Ca 2+ -free and Ca 2+ -bound states (Wang et al, 2008; Akerboom et al, 2009), and high-throughput screening in bacterial colonies (Ibraheem et al, 2011; Zhao et al, 2011a; Akerboom et al, 2012b) and lysates (Tian et al, 2009). A number of engineered variants of GCaMP have been published (Ohkura et al, 2005; Tallini et al, 2006; Tian et al, 2009; Muto et al, 2011); of these the GCaMP5 indicators (Akerboom et al, 2012a) show the best performance in detecting APs in neurons.…”

Genetically Encoded Fluorescent Biosensors for Live-Cell Visualization of Protein Phosphorylation

“…Komatsu et al (2011) developed a backbone with a flexible 116 amino acid linker that improved the dynamic range for a number of kinase and GTPase sensors and was used to rapidly develop several new kinase sensors. Ibraheem et al (2011) used a library approach to optimize linker length, comparing basal and maximally-stimulated states in replica-plated bacterial colonies. This approach allowed rapid assessment of dynamic range in a large number of variants of the probe.…”