Bioinspired Networks of Communicating Synthetic Protocells

Abstract

The bottom-up synthesis of cell-like entities or protocells from inanimate molecules and materials is one of the grand challenges of our time. In the past decade, researchers in the emerging field of bottom-up synthetic biology have developed different protocell models and engineered them to mimic one or more abilities of biological cells, such as information transcription and translation, adhesion, and enzyme-mediated metabolism. Whilst thus far efforts have focused on increasing the biochemical complexity of individual protocells, an emerging challenge in bottom-up synthetic biology is the development of networks of communicating synthetic protocells. The possibility of engineering multi-protocellular systems capable of sending and receiving chemical signals to trigger individual or collective programmed cell-like behaviours or for communicating with living cells and tissues would lead to major scientific breakthroughs with important applications in biotechnology, tissue engineering and regenerative medicine. This mini-review will discuss this new, emerging area of bottom-up synthetic biology and will introduce three types of bioinspired networks of communicating synthetic protocells that have recently emerged.

Keywords: active matter; bottom-up synthetic biology; chemical communication; intelligent material; out-of-equilibrium system; protocell; protocellular material; prototissue.

FIGURE 1

(A) Schematic describing a GOx/HRP enzyme cascade reaction between two populations of coacervate microdroplets. In this system the input signal is represented by glucose (Glc), which gets oxidised to cluconolactone (GlcA) by the GOx-containing coacervate microdroplet. This reaction produces the signalling molecule H ₂ O ₂ , which diffuses radially from the GOx containing coacervate microdroplet and is utilised by the HRP-loaded coacervate microdroplet to oxidise o-PD to 2,3-DAP and produce a fluorescent output signal (Tian et al., 2018). (B) Scheme describing the toehold DNA strand displacement communication mechanism between two proteinosomes. The input chemical signal represented by the single stranded DNA strand ‘A’ diffuses into a proteinosome containing a DNA gate complex and displaces strand Q which is functionalised with a fluorescence quenching moiety. The release of Q causes a fluorescence turn-on of the fluorophore which remains attached to the DNA gate complex inside the proteinosome (Joesaar et al., 2019). (C) Scheme describing quorum sensing in synthetic protocells. Polymer-based protocells contain plasmids coding for T3 RNA polymerase and for the green fluorescent reporter protein (sfGFP). Only at high densities of protocells in dispersion the local concentration of T3 RNA polymerase is high enough to trigger the transcription of sfGFP (Niederholtmeyer et al., 2018). (D) Scheme describing the predatory behaviour of coacervate microdroplets towards proteinosomes. The coacervate “predator” contains sequestered protease K. Electrostatic interactions bring positively-charged coacervate into contact with negatively-charged proteinosome “prey” (1). Protease K digests the protein-polymer material that composes the proteinosome’s membrane and so breaks apart the proteinosome (2). The “sponge-like” nature of the coacervate allows it to take up the membrane components of the proteinosome (3,4) (Qiao et al., 2017). (E) Scheme showing the reactivity of “killer” chitosan polymersomes. The killer polymersomes were loaded with GOx (green shapes) which converted glucose to gluconate “chelator” (red shapes). Gluconate diffused to the Cu²⁺-crosslinked “target” polymersomes and sequestered the copper ions (blue squares), causing the death of the target protocell (Arya et al., 2016). All figures adapted with permission.

FIGURE 2

(A) Schematic of light activated communication within a 3D printed synthetic tissue. Light-activated α-hemolysin gene (LA-αHL) is expressed using in vitro transcription-translation system (IVTT). Once synthesised LA-αHL forms protein pores that allow for the exchange of substrates between protocells of synthetic tissues (Booth et al., 2016). (B) Formation of GUV-based prototissues using a magnetic field on a stainless steel (SS) grid (left). Magnetic GUVs (green round shapes) progressively assemble inside the grid to form prototissues guided by the magnetic field (right) (Li et al., 2020). (C) Scheme explaining the reactivity of prototissues comprising hemi-fused GOx- or HRP-loaded GUVs held together by acoustic pressure fields. Addition of melittin pore protein to this system allows entry of glucose and Amplex Red. Subsequently, glucose is oxidised to gluconolactone inside the GOx-loaded GUV and the signalling molecule H₂O₂ is produced. H₂O₂ diffuses into the other HRP-loaded GUV which catalyses oxidation of Amplex Red to red fluorescent resorufin (Wang et al., 2019). (D) Schematic showing diffusible communication between proteinosomes within a thermoresponsive prototissue spheroid. The input signal glucose (Glc) is converted to gluconolactone (GDL) by GOx inside the green proteinosomes. This reaction also releases the signalling molecule H₂O₂ which diffuses into the red proteinosomes containing HRP. HRP converts ABTS or Amplex red to fluorescent ABTS^•+ or resorufin, respectively (Gobbo et al., 2018). (E) Schematic of chemical communication based on a GOx/HRP enzyme cascade reaction within a protocellular material. Details on the reactivity are reported in (d) (Galanti et al., 2021). (F) Mechanism of the chemical “translation” carried out by protocells for murine stem cells. The scheme shows a representation of the effects of released BDNF on murine stem cells. BDNF is constitutively expressed in the vesicle but cannot diffuse across its membrane. When 3OC6 HSL diffuses into the artificial vesicle, it initiates transcription and subsequent translation of PFO. PFO oligomerises and forms a pore in the membrane of the vesicle. This allows exit of BDNF from the vesicle. BDNF initiates development and branching of murine neural stem cells (Toparlak et al., 2020). (G) Scheme showing a proteinosome (purple sphere) inducing the death of multiple E. coli (green shapes = alive; pink shapes = dead). The positively charged proteinosome surface attracts the negatively charged bacteria. The death of the bacteria is induced through the contact with the ammonium salt present on the proteinosome’s membrane and through the pH-mediated release of chitosan oligosaccharides which are bactericides (Zhao et al., 2020). All figures adapted from original articles with permission.