Roadmap to Building a Cell: An Evolutionary Approach

Abstract

Laboratory synthesis of an elementary biological cell from isolated components may aid in understanding of the fundamental principles of life and will provide a platform for a range of bioengineering and medical applications. In essence, building a cell consists in the integration of cellular modules into system's level functionalities satisfying a definition of life. To achieve this goal, we propose in this perspective to undertake a semi-rational, system's level evolutionary approach. The strategy would require iterative cycles of genetic integration of functional modules, diversification of hereditary information, compartmentalized gene expression, selection/screening, and possibly, assistance from open-ended evolution. We explore the underlying challenges to each of these steps and discuss possible solutions toward the bottom-up construction of an artificial living cell.

Keywords: artificial cell; bottom-up synthetic biology; directed evolution; liposome; synthetic cell; system’s level evolution.

FIGURE 1

Framework to designing a synthetic cell. (A) Building a cell is also a way to question the fundamental principles of life. The diagram depicts our view of the key ingredients constituting a biological cell, and by extension an elementary cell. Cellular unit and environment are entangled and they both constitute the “living system”. We consider information (i.e., the sequence of nucleotides in the DNA and RNA of present-day organisms and viruses) and molecular hardware (i.e., the memory-carrying molecule and the set of proteins, lipids, cofactors, etc, composing the cell) as defining elements of life, but their exact nature is not, as they are both subject to diversity across organisms. In contrast, minimal cellular life can be seen as a set of universal functions that are common to all living things. (B) The molecular hardware is defined as the nature and concentration (C) of each cell’s constituents (x refers to their localization). It differs from one organism to the other and, even, in the modern view of biology, between individual cells from a clonal population. The molecular hardware of a synthetic cell may be a combination of natural parts derived from existing organisms and viruses, and engineering products through in vitro evolution or de novo design. The complexity (or simplicity) of the molecular hardware depends on the composition of the environment. Synthesizing a cell following this approach will yield never-born cells, whose initial conditions supporting life do not exist in nature. (C) Liposome and the key reactions forming the central dogma in biology. (D) Scheme of the in vitro continuous evolution cycle applied to synthetic lipid vesicles. Center, microscopy image of liposomes (colored in magenta) expressing the protein GFP (green) from its gene. *In discontinuous evolution mode, this step would consist of DNA isolation and bulk amplification. **In discontinuous evolution mode, the DNA amplified in bulk is re-encapsulated in new liposomes.

FIGURE 2

Proposed genetic diversification and phenotype interrogation methods for system’s level in vitro evolution of gene-expressing vesicles. (A,B) Combinatorial design and assembly methods (A) Gene assembly and recombination. Homologous recombination in yeast can be performed for gene assembly and recombination of large genetic clusters. (B) Gene order and orientation optimization. Constraint-based combinatorial design as well as in vitro assembly could be employed for gene expression optimization. (C,D) Random mutagenesis methods. (C) Low-fidelity DNA replication. Engineered Φ29 or a similar DNA polymerase can be used for error-prone replication of the entire synthetic genome, thus enabling in-vesicle random mutagenesis. (D) Base editing. Base editor, such as activation-induced deaminase (AID) can be fused to an RNA polymerase, enabling random mutagenesis in both strands of DNA in gene expressing regions. (E,F) Semi-random mutagenesis methods. (E) Programmable base editing. Base editors can be tethered to programmable DNA-binding proteins, which will target random mutagenesis to a narrow region of interest. For this purpose, cytidine or adenine deaminase domains (AID, APOBEC1, TadA) can be fused to MS2 coat protein and tethered to endonuclease-deficient versions of Cas9 (dCas9) or Cpf1 (dCpf1) proteins via MS2-hairpins linked to guide RNA. This flexible architecture would allow a random mutagenesis window of around 100 base pairs (Hess et al., 2016). (F) Error-prone in vitro nick repair. In this proposed method, a nick introduced by a reprogrammable nickase (such as nCas9 or nCpf1) at a region of interest is chewed back with an exonuclease (such as T5 exonuclease), filled in by error-prone DNA polymerase (such as human PolI), and ligated by DNA ligase (such as Taq DNA ligase) to resolve the nick. Such a method would likely constrain the random mutagenesis to up to 200 bases on the targeted strand. (G–I) Screening Methods. (G) Fluorescence-activated cell sorting (FACS). This high-throughput screening method relies on fluorescence measurement of individual particles and sorting by electrostatic deflection. (H) Intelligent image-activated cell sorting (IACS). This high-throughput screening method sorts particles based on their unique morphological features and is based on real-time image-based flow cytometry assisted by artificial intelligence. (I) Dual-photopolymerized microwell array sorting. This image-based single-cell sorting method relies on two-step photopolymerization process: the first to create an array of microwells to capture individual cells and the second to encapsulate undesired cells. The desired cells are removed by washing. (J,K) Selection methods. (J) Compartmentalized partnered replication. When adapted for in vitro, in-liposome use, this approach would rely on linking a phenotype of interest to the expression of a DNA polymerase gene. Differential amplification of a synthetic gene cluster by the expressed DNA polymerase in liposomes would be the basis of selection. (K) Affinity chromatography. Liposomes displaying a molecule of interest can be enriched by specific interaction with a binding partner on a solid support.