16.5 Post-Assembly Molecular Loading

Post-Assembly Molecular Loading describes the incorporation of molecular cargo into a synthetic cell compartment that has already completed boundary closure, requiring cargo to be actively introduced across an already-sealed boundary rather than being passively captured during formation. This route encompasses a range of mechanisms for temporarily or selectively breaching the boundary, exploiting existing transport pathways, or physically delivering cargo, each offering different trade-offs in precision, efficiency, and impact on compartment integrity.

The General Starting Condition

Preformed Compartment Loading

Preformed compartment loading is the general concept of introducing cargo into a compartment that already exists as a fully closed, independent structure, distinguishing this entire category from assembly-time encapsulation, where closure and cargo capture occur together.

Boundary Permeabilization Approaches

Transient Boundary Permeabilization

Transient boundary permeabilization is a loading approach in which the compartment boundary is temporarily made more permeable, allowing cargo to cross that would not normally pass through the intact boundary, followed by a return to the boundary's normal, less permeable state.

Pore-Mediated Molecular Loading

Pore-mediated molecular loading is a loading approach that relies on the temporary formation of discrete pores within the boundary, through which cargo molecules pass into the interior before the pores close again.

Channel-Mediated Molecular Loading

Channel-mediated molecular loading is a loading approach that relies on pre-existing or newly inserted membrane channel proteins to move cargo across the boundary, exploiting a channel's normal transport function to introduce cargo without a general disruption of the boundary.

Carrier-Mediated Molecular Loading

Carrier-mediated molecular loading is a loading approach that relies on membrane transporter proteins to actively move cargo across the boundary, using the transporter's normal conformational transport cycle rather than passive diffusion through an open pore or channel.

Gradient-Driven Molecular Loading

Gradient-driven molecular loading is a loading approach in which a concentration or other physicochemical gradient across the boundary drives net movement of cargo from the external medium into the compartment interior, relying on the gradient itself as the driving force rather than an actively applied external intervention.

Fusion and Direct Delivery Approaches

Fusion-Mediated Cargo Delivery

Fusion-mediated cargo delivery is a loading approach in which a separate carrier compartment, already containing the intended cargo, is merged with the target compartment through vesicle fusion, delivering the cargo into the target's lumen as the two boundaries and interiors combine.

Microinjection-Based Cargo Loading

Microinjection-based cargo loading is a loading approach in which cargo is physically introduced directly into the compartment interior using a fine needle or similar instrument that pierces the boundary, delivering a precisely controlled amount of cargo to a specific target compartment.

Electroporation-Assisted Cargo Loading

Electroporation-assisted cargo loading is a loading approach in which a brief, strong electric pulse is applied to the compartment, transiently creating pores in the boundary through which cargo present in the surrounding medium can enter before the pores reseal.

Mechanical Cargo Introduction

Mechanical cargo introduction is a loading approach in which physical force, such as pressure or shear, is applied to drive cargo across the boundary or to transiently disrupt the boundary sufficiently for cargo to enter.

Restoring and Finalizing the Compartment

Post-Loading Boundary Resealing

Post-loading boundary resealing is the restoration of the compartment boundary to its normal, intact state following a loading approach that involved transient permeabilization, pore formation, or mechanical disruption, relying on the same self-sealing behavior relevant to general boundary integrity.

Post-Loading External Cargo Removal

Post-loading external cargo removal is a processing step in which cargo remaining in the external medium after loading, rather than successfully incorporated into the compartment interior, is separated away, isolating the loaded compartment population from unincorporated cargo.

Completion of the Process

Active Loading Completion

Active loading completion is the point at which a chosen post-assembly loading approach has finished delivering its intended cargo and, where applicable, the boundary has resealed, marking the compartment as ready for subsequent evaluation or use with its newly incorporated cargo in place.