Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

Abstract

Bacterial cellulose (BC) has excellent material properties and can be produced cheaply and sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli. Here, we describe a simple approach for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii was co-cultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which were then colonized by the E. coli upon transfer to selective lysogeny broth media. We show that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, we produced capsules capable of altering their own bulk physical properties through enzyme-induced biomineralization. This novel system, based on autonomous biological fabrication, significantly expands the functionality of BC-based living materials.

Characterizing chemical signaling between engineered “microbial sentinels” in porous microplates

“…Materials differ in their molecular transport properties. Porous hydrogel scaffolds that have been investigated for living materials include agarose (Gerber et al , 2012 ; Gonzalez et al , 2020 ), alginate (Kim et al , 2011 ), acrylamide (Lin et al , 2016 ; Liu et al , 2017 , 2018 ; Sankaran et al , 2019 ), dextran (Guo et al , 2020 ), methacrylates (Liu et al , 2018 ), and bacterial cellulose (Gilbert & Ellis, 2019 ; Birnbaum et al , 2020 ). Cross‐linked agarose has pores that range from 0.5 to 500 µm depending on the fabrication processes, with porosity (the ratio of void volume to total volume of a material) of about 30% (Annabi et al , 2010 ; Rodriguez Corral et al , 2020 ).…”

Synthetic biology as driver for the biologization of materials sciences

“…One way to overcome this limitation would be a combination of ELMs with high-throughput materials characterization. For example, ELM-producing micro-organisms could be mutated and cultivated in a format where each cell would produce one distinct ELM [ 348 ] that could subsequently be screened for desired materials properties using high-throughput robotics for measuring, for example, rheological properties [ 349 ]. The establishment of such directed evolution would allow evolving materials synthesized from renewable sources toward any property or function that can be synthesized by any engineered cell or multicellular community.…”

Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

“…In E. coli these genes are encoded on two divergent operons, however a number of landmark ELM studies have elegantly functionalised curli by rearranging these genes into an easier-to-engineer synthetic linear operon (Kan et al, 2019). The linear operon format enables researchers to more readily modify curli through C-terminal CsgA fusions (small peptides or whole proteins) or by exploiting the signal peptide (the first 22 amino acids of CsgA) to secrete heterologous proteins through the Curli T8SS (Assalkhou et al, 2007;Birnbaum et al, 2020;Botyanszki et al, 2015;Nguyen et al, 2014;Nussbaumer et al, 2017;Seker et al, 2017;Sivanathan and Hochschild, 2012;Tay et al, 2017;Van Gerven et al, 2014;Wang et al, 2017;Zhong et al, 2014).…”

“…As a case-study, we used KTK cloning to express E. coli's Curli system, which produces the best-studied bacterial amyloid; a polymeric protein structurally rich in betasheets that forms a fibre with exceptional strength, stability and resistance (Evans and Chapman, 2014). Curli has been a focus for many ELM-associated studies (Birnbaum et al, 2020;Chen et al, 2014;Nguyen et al, 2014;Seker et al, 2017), and offers promise for making a novel composite with BC in cells that can co-produce both materials. The Curli production system is also representative of a Type VIII secretion system (T8SS) and so its expression offers a tractable solution for protein secretion from BC-producing cells.…”