Biology subjects

Molinari, S.

Publications and source records attributed to Molinari, S..

7 recordsLinked to original sources

bioRxiv2025

Engineered Living Materials for Bioproduction Downstream Processing

De novo engineered living materials (ELMs) are cellular systems that self-assemble into macroscopic structures through genetically encoded interactions, offering a route to programmable materials grown directly from living cells. Despite their promise, the molecular design principles that enable scalable self-assembly in de novo ELMs remain poorly understood. Here, we engineer Escherichia coli to display elastin-like polypeptides (ELPs) on the outer membrane, transforming single cells into self-assembling living materials through weak intermolecular interactions. By tuning the polarity of ELP sequences, we generate assemblies spanning micrometer- to centimeter-length scales that sediment within a few hours while preserving cellular metabolic activity. We demonstrate the portability of this platform across genetic backgrounds and inducible expression systems, and deploy it in an ethanologenic E. coli strain as a proof of principle. In small-scale fermentation settings, ELP-based ELMs enable controllable flocculation, reduce filtration time by more than threefold, and maintain ethanol production performance comparable to that of the parental strain. Together, this work establishes ELP surface display as a modular strategy for constructing de novo engineered living materials and defines initial genetic design rules linking molecular-scale interactions to emergent macroscopic organization.

synthetic biology↗

bioRxiv2025

Bacterial extracellular vesicles as a tunable platform for vaginal drug delivery

There is a critical gap in the development of new therapeutic platforms designed to treat gynecologic and obstetric diseases. Compared to systemic drug delivery, vaginal drug administration of nanoparticle formulations limits off-target side effects while increasing therapeutic concentration in target tissues, showing promise for clinical translation. However, these formulations suffer from limited scalability, high-cost reagents, and long optimization timelines. Recent work highlights the potential of bacterial extracellular vesicles (bEVs) as a low-cost, tunable platform for therapeutic applications. Here, we evaluate bEVs as a therapeutic carrier for vaginal drug delivery. We demonstrate the loading of the model protein moxNeonGreen into Escherichia coli Nissle 1917 bEVs. By optimizing growth parameters, we increase protein loading into bEVs. We evaluate the effect of bEVs on the vaginal microenvironment, and observe no negative impact on vaginal epithelial cells, endocervical cells, or vaginal bacteria in vitro. Additionally, we observe the retention of bEVs in the murine female reproductive tract for more than six hours. This study provides a framework for using genetically engineered bEVs to rapidly generate customizable therapies for a range of gynecologic and obstetric conditions, addressing longstanding challenges in womens health therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/658669v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e483d2org.highwire.dtl.DTLVardef@8a6d2corg.highwire.dtl.DTLVardef@5756borg.highwire.dtl.DTLVardef@5fdb67_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Tunable dynamics in a multi-straintranscriptional pulse generator

A major challenge in synthetic biology is the manipulation of engineered gene circuits toward a specified behavior. This challenge becomes more difficult as synthetic systems become more complex by incorporating additional genes or strains. Here we demonstrate that circuit dynamics can be tuned in synthetic consortia through the manipulation of strain fractions within the community. To do this, we constructed a microbial consortium comprised of three strains of engineered Escherichia coli that, when co-cultured, use homoserine lactone (HSL) mediated intercellular signaling to create a multi-strain incoherent type-1 feedforward loop (I1-FFL). Like naturally occurring I1-FFL motifs in gene networks, this engineered microbial consortium acts as a pulse generator of gene expression. We demonstrated that the amplitude of the pulse can be easily tuned by adjusting the relative population fractions of the strains. We created a mathematical model for the temporal dynamics of the microbial consortium and, using this model, identified population fractions that produced desired pulse characteristics. Our work demonstrates that intercellular gene circuits can be effectively tuned simply by adjusting the starting fractions of each strain type.

synthetic biology↗

bioRxiv2021

A de novo matrix for macroscopic living materials from bacteria

Summary ParagraphEngineered living materials (ELMs) are composites of living cells embedded in a biopolymer matrix that combine the desirable properties of natural biomaterials with non-natural, tailored properties. ELMs with a wide range of sophisticated biological functions have been created by engineering the embedded cells using synthetic biology. Engineering a de novo biomolecular matrix would offer control over material assembly, structure, and composition, thus enabling us to grow macroscopic ELMs with customizable mechanical properties. However, we have lacked the genetic tools and design rules to genetically encode a synthetic matrix that programs collective cell self-organization into macroscopic structures. Here we report growth of macroscopic ELMs from Caulobacter crescentus cells that display and secrete an engineered self-interacting protein. This protein formed an extracellular de novo matrix and assembled cells into hierarchically-ordered, centimeter-scale ELMs. We showed that the mechanical, catalytic, and morphological properties of these ELMs can be tuned through genetic modification of the matrix. Our work identifies novel genetic tools, design and assembly rules for growing macroscopic ELMs with both wide-ranging mechanical properties and customizable functions. We anticipate the modularity of this approach will permit the incorporation of different protein polymers in the de novo matrix, thus allowing to generate ELMs with a variety of desired structures and compositions of the bulk material. We envision specific matrix properties that can be combined synergistically with existing cellular functions to greatly expand the opportunities for ELMs in human health, energy, and the environment.

synthetic biology↗

bioRxiv2020

Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions, but require exogenously added material or have limited programmability. Here we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or resilin-like polypeptide, demonstrating this system is flexible. The SC(-)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards the formation of cohesive engineered living materials. IMPORTANCEEngineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy intensive processing or have limited tunability. We propose a bacterially-synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

synthetic biology↗

bioRxiv2019

Molecular signaling involved in immune system activation against root-knot nematodes by bio-control agents in tomato plants

The expression of key defense genes was detected in roots and leaves of tomato plants until the 12th day after treatments with a mixture of beneficial bio-control agents (BCAs), as soil-drenches. The expression of the same genes was monitored in pretreated plants at the 3rd and 7th day since the inoculation with the root-knot nematode Meloidogyne incognita. Genes dependent on SA-signaling, such as the Pathogenesis Related Genes, PR1, PR3, and PR5, were systemically over-expressed at the earliest stages of BCA-root interaction. BCA pre-treatment primed plants against root-knot nematodes. The expression of PR-genes and of the gene encoding for the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO), which catalyzes the last step of ethylene biosynthesis, was systemically enhanced after nematode inoculation in primed plants. Defense related enzyme activities, such as endochitinase and glucanase, were higher in roots of BCA-treated than in those of untreated plants, as well. On the contrary, the expression of genes dependent on JA/ET-signaling, such as Jasmonate Ethylene Response Factor 3 (JERF3), did not increase after nematode inoculation in primed plants. The antioxidant system, as indicated by catalase gene expression and ascorbate peroxidase activity, was repressed in infected colonized roots. Therefore, Systemic Acquired Resistance (SAR), and not Induced Systemic Resistance (ISR), is proposed as the molecular signaling that is activated by BCA priming at the earliest stages of root-nematode interaction. Such BCA-induced activation of the plant immune system did not directly act against nematode motile juveniles penetrating and moving inside the roots. It resulted in a drastically decreased number of sedentary individuals and, then, in an augmented ability of the plants to contrast feeding site building by invasive juveniles.

plant biology↗

bioRxiv2018

Synthetic pluripotent bacterial stem cells

A defining property of stem cells is their ability to differentiate via asymmetric cell division, in which a stem cell creates a differentiated daughter cell but retains its own phenotype. Here, we describe a synthetic genetic circuit for controlling asymmetrical cell division in Escherichia coli. Specifically, we engineered an inducible system that can bind and segregate plasmid DNA to a single position in the cell. Upon division, the co-localized plasmids are kept by one and only one of the daughter cells. The other daughter cell receives no plasmid DNA and is hence irreversibly differentiated from its sibling. In this way, we achieved asymmetric cell division though asymmetric plasmid partitioning. We also characterized an orthogonal inducible circuit that enables the simultaneous asymmetric partitioning of two plasmid species - resulting in pluripotent cells that have four distinct differentiated states. These results point the way towards engineering multicellular systems from prokaryotic hosts.

synthetic biology↗