DNA Microgels as a Platform for Cell-Free Protein Expression and Display

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired. traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microger formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene,sets, and protein display. We utilize mictogels to show successful protein production and capture of a model protein, green fluorescent protein, (GFP), which isjurther used to demonstrate a successful gene enrichment through-fluorescence-activated cell sorting (PACS) of a mixed population of rnicrogels containing4he GFP gene. Through psoralen cross-linking of the hydrogels, have synthesized DNA microgels capable of Surviving denatufing conditions while still possessing the ability to produce protein. Lastly, we demonstrate- a method of producing extremely high local gene- concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 mu m in diameter. These DNA gels can serve as a novel cell free platform for integrated protein expression and :display, which-can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and' limitations.