Linking Genotype and Phenotype in Protein Synthesizing Liposomes with External Supply of Resources

Abstract

Reconstituting an elementary gene expression system inside self-assembled lipid vesicles to mimic the cellular synthesis machinery is at the core of the development of a minimal cell following a bottom-up synthetic biology approach. The ability to operate the expression of multiple genes in a controlled manner and to generate the output proteins with predictable dynamics in liposomes relies on the link between genotype and phenotype. Here, we established this link in surface-tethered liposomes producing proteins from a linear DNA template using a reconstituted transcription/translation/aminoacylation apparatus fuelled by external supply of feedstock. The amounts of entrapped DNA molecules and synthesized proteins were visualized by fluorescence confocal microscopy in individual vesicles. We showed that there exists no linear correlation between the amount of encapsulated genes and the level of output proteins, which is a consequence of the compositional heterogeneity between liposomes due to the low-copy number of some constituents, as well as interfacing differences with the nutrient-containing environment. In order to decouple gene activity from those sources of variability and, thus, infer the probabilistic occupancy of transcriptionally active genes in protein synthesizing liposomes, we developed a dual gene expression assay consisting of the production of two fluorescent reporter proteins of distinguishable colors from two different DNA templates. The stochastic color-coding of the vesicles was analyzed and compared to the color pattern expected from a Poisson distribution of encapsulated genes. Unexpectedly, we found that the apparent number of transcriptionally active DNA molecules in liposomes corresponds only to ca. 10% of the bulk concentration. We believe that our study provides new insights about the relationship between the genotype and phenotype in protein synthesizing liposomes, which is of primary importance toward the construction of a programmable artificial cell implemented with regulatory gene networks of predictable dynamics.

Spontaneous Structuration in Coacervate‐Based Protocells by Polyoxometalate‐Mediated Membrane Assembly

“…Compartmentalization is a necessary and overarching attribute of all living systems and is increasingly recognised as an essential paradigm of synthetic cellularity, a key step in the origin of life and an important consideration for developing new approaches to novel functional ensembles . Strategies towards membrane‐delineated compartmentalization have primarily focused on the use of a diverse range of amphiphilic building blocks that undergo spontaneous or directed assembly in aqueous solutions or oil/water mixtures to produce micro‐compartments such as fatty acid vesicles and liposomes, polymersomes, poly­electrolyte capsules and capsosomes, colloidosomes, protein‐based capsules and proteinosomes . In most cases, these micro‐architectures comprise a single water‐filled micrometre‐sized interior enclosed by a semi‐permeable membrane capable of selective binding, uptake and retention of functional components such as inorganic nanoparticles and enzymes.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…A successful example where such interaction could be established is surface‐tethered liposomes which can produce proteins from a template linear DNA through a reconstructed transcription/translation/aminoacylation setup powered by external feedstock source. Further, fluorescence confocal microscopy revealed accumulation of entrapped DNA molecules and produced proteins in single vesicles (Nourian & Danelon, 2013). Likewise, synthetic minimal cells (synells) are engineered, which include genetic circuits, to embrace multiple‐part genetic cascades which could be adjusted through signals received from the outside environment and interliposomal communication.…”

Construction de cellules synthétiques

“…L'encapsulation de ces réactions complexes est compliquée. À cela s'ajoute l'hétérogénéité de l'encapsulation, et donc de l'expression, d'un liposome à l'autre, quelle que soit la méthode utilisée [52,53]. Au-delà de l'encapsulation, la construction d'une membrane active est un défi.…”