Browsing by Author "Molinari, Sara"

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    A de novo matrix for macroscopic living materials from bacteria
    (Springer Nature, 2022) Molinari, Sara; Tesoriero, Robert F.; Li, Dong; Sridhar, Swetha; Cai, Rong; Soman, Jayashree; Ryan, Kathleen R.; Ashby, Paul D.; Ajo-Franklin, Caroline M.
    Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.
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    A synthetic system for asymmetric cell division in Escherichia coli
    (2019-07-16) Molinari, Sara; Bennett, Matthew R.; Wagner, Daniel S.
    One 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, I describe a synthetic genetic circuit for controlling asymmetric cell division in E. coli in which a progenitor cell creates a differentiated daughter cell while retaining its original phenotype. Specifically, an inducible system was engineered that can bind and segregate plasmid DNA to a single position in the cell. Upon division, the colocalized 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, asymmetric cell division happens though asymmetric plasmid partitioning. This system was further used to achieve physical separation of genetically distinct cells by tying motility to differentiation. Finally, an orthogonal inducible circuit was characterized 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.
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    Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
    (American Society for Microbiology, 2021) Orozco-Hidalgo, Maria Teresa; Charrier, Marimikel; Tjahjono, Nicholas; Tesoriero, Robert F.; Li, Dong; Molinari, Sara; Ryan, Kathleen R.; Ashby, Paul D.; Rad, Behzad; Ajo-Franklin, Caroline M.
    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 an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a 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 (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered 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 toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.