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

• Main Authors: Ibraheem Andreas, Yap Hongkin, Ding Yidan, Campbell Robert E
• Format: Article
• Language: English
• Published: BMC 2011-11-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/11/105

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Escherichia coli</it>. 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.</p> <p>Conclusion</p> <p>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%).</p>