Transport Subsystem
The transport subsystem of a synthetic cell is responsible for moving materials across the cell membrane — either passively or actively, and with varying degrees of selectivity. Transport is a prerequisite for nearly every other subsystem: the Metabolic Subsystem requires nutrient import and waste export, the Sensing Subsystem must detect external signals that may not cross the membrane unaided, and the Communications Subsystem relies on controlled molecular exchange between cells.
Transport mechanisms
Synthetic cell membranes are formed from lipid bilayers or polymersomes that are intrinsically impermeable to most molecules larger than water and small gases. Achieving selective permeability requires the incorporation of protein channels or other transport elements into the membrane.
Pore-forming proteins
The most widely used transport element in synthetic cells is α-hemolysin, a bacterial pore-forming protein that self-assembles into heptameric channels in lipid bilayers. Once inserted, α-hemolysin pores allow passive diffusion of small molecules (up to approximately 3 kDa) across the membrane, including nucleotides, amino acids, small signaling molecules such as IPTG, and fluorescent reporters. Because the pores are non-selective within this size range, α-hemolysin is primarily used where broad permeability to small molecules is desired — for example, to allow continuous feeding of substrates from an external buffer (see Metabolic Subsystem) or to enable release of encapsulated signals to neighboring cells (see Communications Subsystem).
Controlled pore formation
A more sophisticated approach is to control when pores form, using membrane composition or external signals to gate transport. This converts the transport subsystem from a passive channel into an active, logic-capable interface.
Demonstration: Membrane AND gate for controlled secretion (Hilburger et al., 2019)
Hilburger and colleagues at Northwestern demonstrated artificial cells capable of controlled secretion using a membrane-based AND gate[1]. The system used giant unilamellar vesicles (GUVs) containing α-hemolysin monomers; pore assembly — and hence cargo release — required both α-hemolysin AND oleic acid to be present. In the absence of oleic acid, the membrane composition prevented α-hemolysin from assembling into functional heptameric channels, keeping the membrane impermeable. When oleic acid was added externally via micelles, it incorporated into the bilayer, changing its composition and enabling pore formation and release of encapsulated cargo. This demonstrated that membrane-based Boolean logic could complement genetic circuits and provided a new method for temporal control of vesicle permeability through protein–lipid interactions.
External activation of transport
Rather than relying on diffusible chemical signals to control pore formation, several groups have demonstrated transport control using physical stimuli — mechanical force, magnetic fields, or light — applied from outside the synthetic cell. These approaches are attractive because the activation signal does not need to cross the membrane and does not interfere with internal biochemistry.
Demonstration: Mechanosensitive channels (Majumder et al., 2017)
Majumder and colleagues demonstrated liposomes containing cell-free transcription–translation systems expressing both the mechanosensitive ion channel MscL and a calcium biosensor[2]. MscL opens in response to membrane tension, providing a direct pathway from mechanical inputs to molecular transport and downstream biosensor readout.
Demonstration: Light-activated pore formation (Booth et al., 2016)
Booth and colleagues demonstrated light-activated production of α-hemolysin pores in droplet-based synthetic cells, enabling directional and spatially selective transport across engineered bilayer interfaces[3]. By illuminating specific regions of a synthetic tissue, pore formation — and hence molecular transport and electrical communication — could be restricted to selected interfaces.
Transport in the control architecture
The transport subsystem sits at the boundary between the synthetic cell interior and its environment, interfacing with nearly every other subsystem:
- Import: nutrients, energy substrates, and signaling molecules must enter the cell through the membrane. The selectivity and rate of import constrain what the Metabolic Subsystem and Sensing Subsystem can access.
- Export: waste products (inorganic phosphate, ADP) must be removed to prevent inhibition of internal processes; signaling molecules must be released to communicate with neighboring cells.
- Gating: controlled transport — triggered by chemical, mechanical, magnetic, or optical signals — converts the membrane from a passive barrier into an active computational element that can implement logic, timing, and spatial selectivity.
References
- ↑ C. E. Hilburger, M. L. Jacobs, K. R. Lewis, J. A. Peruzzi, and N. P. Kamat, Controlling secretion in artificial cells with a membrane AND gate. ACS Synthetic Biology 8(6):1224–1230, 2019. DOI: 10.1021/acssynbio.8b00435
- ↑ S. Majumder, J. Garamella, Y. L. Wang, M. DeNies, V. Noireaux, and A. P. Liu, Cell-sized mechanosensitive and biosensing compartment programmed with DNA. Chemical Communications 53(53):7349–7352, 2017.
- ↑ M. J. Booth, V. R. Schild, A. D. Graham, S. N. Olof, and H. Bayley, Light-activated communication in synthetic tissues. Science Advances 2(4):e1600056, 2016.