Presentation Information
[P04-589]Barcode-resolved microfluidic screening for high-throughput biosensor evolution
○Svenja Lützow1, Harrison Steel1 (1. University of Oxford (UK))
Keywords:
synthetic biology,directed evolution,microfluidics,biosensors
Directed evolution enables the discovery and optimization of proteins and genetic circuits without requiring detailed knowledge of sequence–function relationships. By iteratively introducing mutations and selecting improved variants, this approach has been used to develop systems with novel activities and therapeutic potential. To evaluate large mutant libraries, image-based pooled screens offer a powerful strategy: they combine single-cell resolution with high-throughput optical readouts, directly linking genotype to phenotype. My project will apply this principle to barcoded biosensor libraries, where each protein variant is associated with a unique DNA barcode, enabling scalable mapping of sequence to function.
As a proof of concept, I have implemented the sequential FISH process to detect barcoded constructs encoding different fluorescent proteins. Key steps have been demonstrated, including maintenance of cell viability, fixation, and some probe-based detection of barcodes on a flow cell platform. The next stage is to refine the FISH workflow on the mother machine and apply it to a small protein library such as magnetic protein variants. An advanced automated liquid-handling system will be integrated with the microfluidic platform, increasing throughput and reproducibility and enabling the eventual application of this approach to larger protein libraries and biosensing targets such as nanobody-based sensors. This framework has the potential to uniquely identify thousands of protein variants by leveraging modular 14-bit barcodes detected across multiple hybridization rounds, opening the door to scalable, image-based directed evolution.
As a proof of concept, I have implemented the sequential FISH process to detect barcoded constructs encoding different fluorescent proteins. Key steps have been demonstrated, including maintenance of cell viability, fixation, and some probe-based detection of barcodes on a flow cell platform. The next stage is to refine the FISH workflow on the mother machine and apply it to a small protein library such as magnetic protein variants. An advanced automated liquid-handling system will be integrated with the microfluidic platform, increasing throughput and reproducibility and enabling the eventual application of this approach to larger protein libraries and biosensing targets such as nanobody-based sensors. This framework has the potential to uniquely identify thousands of protein variants by leveraging modular 14-bit barcodes detected across multiple hybridization rounds, opening the door to scalable, image-based directed evolution.
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