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

Abstract

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.

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Micro‐compartmentalizing DNA templates, for example, DNA brushes (Karzbrun, Tayar, Noireaux, & Bar‐Ziv, 2014) and DNA gel (Kahn et al, 2016), as opposed to microencapsulation, enabled consistent protein expression when replenished with nutrients and energy, making such platform practical for the studies of synthetic networks (Zhou, Wu, Cui, Lai, & Zheng, 2018). Such life‐like systems, while sustaining pliability and subjugation of the constituents for demanded objectives could eliminate inessential aspects of the concurrent cells (Martini & Mansy, 2011).…”

How to Construct DNA Hydrogels for Environmental Applications: Advanced Water Treatment and Environmental Analysis

“…Following RCA and MCA, extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels with a diameter of 1–2 µm were produced. Noteworthily, chemical cross‐linking of psoralen into the hydrogels endowed them with the capacity of withstanding extreme conditions, which will absolutely boost real application . The hydrogel system provides a new platform for producing protein, solving the matter of stability in real environment, to some extent.…”

Communicating artificial cells

“…Although the component parts do not have to be those found in natural cells, artificial cells are typically made from biological molecules. For example, the compartment that houses the cellular mimic usually consists of phospholipid vesicles, even though compartments can be built from proteins, inorganic particles, block copolymers, polymers that form hydrogel structures, and submillimeter scale recesses within silicon chips [27][28][29][30][31]. Lipid bilayers that separate internal and external aqueous solutions are desirable, because such structures are better able to exchange molecules with the environment and can more easily accommodate components that confer molecular specificity, such as membrane proteins.…”