Permeability‐Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems

Abstract

In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is challenging. Here, a facile strategy is presented to build permeability‐configurable compartments, and marked advantages of such compartmentalization are shown in reconstituting sustained synthetic biology systems in vitro. Through microfluidics, the strategy produces micrometer‐sized layered microgels whose shell layer serves as a sieving structure for biomolecules and particles. In this configuration, the transport of DNAs, proteins, and bacteriophages across the compartments can be controlled an guided by a physical model. Through permeability engineering, a compartmentalized cell‐free protein synthesis system sustains multicycle protein production; ≈100 000 compartments are repeatedly used in a five‐cycle synthesis, featuring a yield of 2.2 mg mL−1. Further, the engineered bacteria‐enclosing compartments possess near‐perfect phage resistance and enhanced environmental fitness. In a complex river silt environment, compartmentalized whole‐cell biosensors show maintained activity throughout the 32 h pollutant monitoring. It is anticipated that permeability‐engineered compartmentalization should pave the way for practical synthetic biology applications such as green bioproduction, environmental sensing, and bacteria‐based therapeutics.

Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets

“…Semi-permeable hydrogels [ 28 ] or microcapsules [ 29 ] are another state-of-the-art approach for controlling the biochemical reactions within micro-compartments. In these systems, the different reagents smaller than the pore size can diffuse into micro-compartments.…”

“…The peptide concentration increased only slightly to 34 nM when the incubation was continued for a total of 24 h (Figure S3), which suggested that the peptide delivery was mostly completed in the first 3 h. These results demonstrated the feasibility of using nanodroplets to achieve delivery of medium-sized biomolecules into water-in-fluorinated-oil microdroplets. Semi-permeable hydrogels [28] or microcapsules [29] are another state-of-the-art approach for controlling the biochemical reactions within micro-compartments. In these systems, the different reagents smaller than the pore size can diffuse into micro-compartments.…”

AI-driven high-throughput droplet screening of cell-free gene expression

“…CFE systems, particularly those based on cell lysates, represent promising platforms for various synthetic biology applications, including therapeutic development 8 , genetic part/circuit prototyping biomanufacturing 7,24 , artificial cell construction 25,26 , natural product biosynthesis [27][28][29] , and biomanufacturing 30,31 . Central to these applications is the need for consistent and efficient protein expression.…”

Applications of Serine Integrases in Synthetic Biology over the Past Decade

“…Relying on the supports of computer science, serine integrases and their att sites could be designed, analyzed, and evolved [121] as variants with diverse properties (e.g., affinity [17,106,122], catalytic efficiency [106,[123][124][125][126], specificity [12,120,127], orthogonality [124,128], and chimerism [129]). Furthermore, other technologies including cell-free protein synthesis [130], microfluidics [131], and artificial cells [132,133] will support the research of serine integrases (or tyrosine recombinases) in different synthetic biology applications.…”