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 microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing 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 μm 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.
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Jason S
JS
Kahn
Department of Biomedical Engineering, Cornell University , Ithaca, New York 14853, United States.
Roanna C H
RC
Ruiz
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Swati
S
Sureka
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Songming
S
Peng
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Thomas L
TL
Derrien
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Duo
D
An
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.|Kavli Institute at Cornell for Nanoscale Science, Cornell University , Ithaca, New York 14853, United States.
Dan
D
Luo
D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005363:Ficusin / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D020100:Hydrogels / Q000138:chemical synthesis / Q000737:chemistry*; D010957:Plasmids; D014176:Protein Biosynthesis / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics*
D003432:Cross-Linking Reagents; D004129:Dimethylpolysiloxanes; D020100:Hydrogels; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C013830:baysilon; D004247:DNA; D005363:Ficusin