(1036-A) Colony Picking to Directed Evolution: High-Throughput Functional Screening with Hollow Hydrogel Microparticles

Abstract: Cells are essential for discovering and producing functional proteins tailored for life science, diagnostic, and therapeutic uses. However, there is an unmet need for high-throughput screening tools capable of analyzing large clone libraries and performing functional assessment and selection. Traditional workflows such as agar plates or microtiter plates fall short in screening capacity and functional evaluation, lacking the required resolution to efficiently select variants of interest. Robotic colony pickers and automated liquid handlers also remain tethered to the limitations of microtiter plates.
To address these challenges, we have introduced PicoShells, picolitre scale hollow microparticles that enable compartmentalization, culture, and phenotypic screening and sorting of clones via fluorescence activated cell sorting (FACS). Their semi-permeable design facilitates nutrient and analyte exchange while retaining large proteins and DNA, preserving the crucial phenotype-genotype link within each PicoShell. Compared to single bacterial cell sorting via FACS, growing colonies in PicoShells amplify the signal-to-noise ratio for clonal analysis, mitigating the noise inherent in single-cell fluorescence, thereby improving selection efficiency and purity. PicoShells can be seamlessly integrated into any cell screening or directed evolution workflow for screening of millions of clones, performing multi-step assays, from lysis to specific assay condition exposures, facilitating functional analysis and sorting via standard FACS instruments.
We demonstrate PicoShells in the directed evolution and high-throughput screening of E. coli expressing a calcium biosensor, GCaMP. In a proof-of-concept, we mixed E. coli expressing calcium-insensitive EGFP with calcium-sensitive GCaMP at equal ratios. Using FACS, we sorted the top 15% of clones that exhibited maximum fluorescence in calcium-rich media. Subsequently, by switching to a calcium-depleted medium, we distinguished between calcium-responsive and non-responsive strains and performed a second sort. These target clones, exhibiting high fluorescence with calcium and reduced fluorescence without calcium, were sequenced, achieving a 98% purity in recovering GCaMP genes in a single round of selection. Building on this success, we are screening a 1024-variant GCaMP library, generated by mutating the N-linker position between the circularly permuted GFP and the M13 peptide using site saturation mutagenesis. While traditional methods might take weeks to screen libraries of this size, using PicoShells combined with FACS we are analyzing over a million clones daily. Our objective is to comprehensively assess the functionality of all variants, identifying those with enhanced sensitivity and a vast dynamic range, underscoring PicoShell's ability to screen diverse pooled libraries for screening and directed evolution.
PicoShells are a powerful tool for high-throughput screening, linking functional activity of cell-produced proteins to underlying DNA. As demonstrated by the screening of calcium-sensitive fluorescent biosensors, this technology has the potential to accelerate colony screening and directed evolution research paving the way for groundbreaking discoveries and advancements in protein and cell engineering.