Cell mimicry: bottom-up engineering of life

Biology is well equipped in exploiting a large number of out of equilibrium processes to support life. A complete understanding of these mechanisms is still in its infancy due to the complexity and number of the individual components involved in the reactions. However, a bottom up approach allows us to replicate key biological processes using a small number of basic building blocks. Moreover, this methodology has the added advantage that properties and characteristics of the artificial cell can be readily tuned and adapted. 

Here, I will present strategies for the design and synthesis of artificial cells based on hydrophobic effects such as lipid vesicles and proteinosomes and liquid-liquid phase separation of oppositely charged components (coacervates) and describe how these compartments may be used as platforms for implementing dynamic biological behaviour. 

Bottom-up synthetic biology aims to substitute for missing/lost cellular activity or to add non-native function to mammalian cells and tissue. We focus on hydrogel-based artificial cells equipped with a specific life-like function, in particular, energy generation and cellular communication. 

The creation of self-sustaining systems require energy generation. In mammalian cells, mitochondria are responsible for the chemically driven adenosine triphosphate (ATP) synthesis. Here, mitochondria are used as a natural ATP producing subunit in hydrogel-based artificial cells. We co-encapsulated the ATP-dependent enzyme firefly luciferase with purified mitochondria to demonstrate the utilization of mitochondrially produced ATP in a cell-sized carrier, exemplified by the bioluminescent decarboxylation of D-luciferin. 

Collective behaviour in multicellular assemblies and on tissue level requires communication. Consequently, the integration of artificial cells with mammalian cells would benefit from supportive signal exchange between the artificial and natural entity. First, a one-way signal transfer is established by equipping two different types of artificial cells with catalytic function using metalloporphyrins as artificial enzymes that mimic cytochrome P450 enzyme (CYP450) activity. Second, artificial cells that can eavesdrop on mammalian cells in 2D and 3D are demonstrated. 

Membranes play a vital role in a variety of physiological processes. Recapitulating these processes outside of the cell may allow us to better understand them as well as design an entirely new class of materials that can sense, transport, or target important biological signals and molecules. In this talk, I will present our recent work using model membranes (ex. liposomes and polymersomes) and cell-free expression systems to (1) uncover the role of membrane mechanical properties on the folding of model membrane proteins and (2) design a membrane-based nanoparticle for biosensing applications. First, I will describe how membrane physical properties, such as elasticity and hydrophobic thickness, impacts the folding, location, and integration of model membrane proteins. Next, I will describe our recent work to demonstrate how compartmentalization of cell-free extracts in membranes provides new capabilities for cell-free biosensing. Our approach, bridging synthetic biology techniques and model membrane assembly, provides an innovative yet simple method to probe the role of membrane composition and biophysical properties on protein dynamics and to advance the design of drug delivery carriers. 

A major goal of synthetic biology is to understand the transition between non-living matter and life. The bottom-up development of an artificial cell would provide a minimal system with which to study the border between chemistry and biology. So far, a fully synthetic cell has remained elusive but chemists are progressing towards this goal by reconstructing cellular subsystems. Cell boundaries, likely in the form of lipid membranes, were necessary for the emergence of life. In addition to providing a protective barrier between cellular cargo and the external environment, lipid compartments maintain homeostasis with other subsystems to regulate cellular processes. Our lab is exploring different chemical approaches for making cell-mimetic compartments. Synthetic strategies to drive membrane formation and function, including bioorthogonal ligations, dissipative self-assembly, and reconstitution of biochemical pathways, will be discussed. Chemical strategies aim to recreate the interactions between lipid membranes, the external environment, and internal biomolecules, and will clarify our understanding of life at the interface of chemistry and biology.

Medicine is taking its first steps towards patient-specific cancer care. Nanoparticles have many potential benefits for treating cancer, including the ability to transport complex molecular cargoes including siRNA and protein, as well as targeting to specific cell populations. 

The talk will discuss ‘barcoded nanoparticles’ that target sites of cancer where they perform a programmed therapeutic task. Specifically, liposomes that diagnose the tumour and metastasis for their sensitivity to different medications, providing patient-specific drug activity information that can be used to improve the medication choice. 

The talk will also describe how liposomes can be used for degrading the pancreatic stroma to allow subsequent drug penetration into pancreatic adenocarcinoma, and how nanoparticle’ biodistribution and anti-cancer efficacy is impacted by patient’ sex and more specifically, the menstrual cycle. 

The evolution of drug delivery systems into synthetic cells, programmed nanoparticles that have an autonomous capacity to synthesize diagnostic and therapeutic proteins inside the body, and their promise for treating cancer and immunotherapy, will be discussed.

Living cells take advantage of spontaneous aggregation of certain proteins and RNA species to spatially and dynamically organize a variety of molecules and reactions. Taking inspiration from nature, our group is developing methods to build assemblies, condensates and patterns using structured motifs built with artificial DNA, that are relevant to the synthesis of self-regulated, autonomous biomaterials. I will present our recent results on controlling the rate of self-assembly and condensation and dissolution of artificial DNA motifs using chemical reactions. I will also discuss mathematical models that support and guide our experiments by capturing specific and non-specific interactions among the motifs.

The evolution of cellular compartments for spatially and temporally controlled assembly of biological processes was an essential step in developing life by evolution. Synthetic approaches to cellular-like compartments are still lacking well-controlled functionalities, as would be needed for more complex synthetic cells. With the ultimate aim to construct life-like materials such as a living cell, matter-to-life strives to reconstitute cellular phenomena in vitro – disentangled from the complex environment of a cell. In recent years, working towards this ambitious goal gave new insights into the mechanisms governing life. With the fast-growing library of functional modules assembled for synthetic cells, their classification and integration become increasingly important. We will discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature’s own prototype. Particularly, we will focus on large outer compartments, complex endomembrane systems with organelles and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two highly promising technologies which can achieve the integration of these functional modules into sophisticated multifunctional synthetic cells. 

Synthetic biology (SB) is one of the most promising fields of research for the 21st century. SB offers powerful new ways to improve human health, build the global economy, manufacture sustainable materials, and address climate change. However, current access to SB-enabled breakthroughs is unequal, largely due to bottlenecks in infrastructure and education. Here, I describe our efforts to re-think the way we engineer biology using cell-free systems to address these bottlenecks. We show how the ability to readily store, distribute, and activate low-cost, freeze-dried cell-free systems by simply adding water has opened new opportunities for on-demand biomanufacturing of vaccines for global health, point-of-care diagnostics for environmental safety, and education for SB literacy and citizenship. By integrating cell-free systems with artificial intelligence (AI), we also show the ability to accelerate the production of carbon-negative platform chemicals. Looking forward, advances in engineering tools and new knowledge underpinning the fundamental science of living matter will ensure that SB helps solve humanity’s most pressing challenges.