ABSTRACT
Engineered microbial communities show promise in a wide range of applications, including environmental remediation, microbiome engineering, and synthesis of fine chemicals. Here we present methods by which bacterial aggregates can be directed into several distinct architectures by inducible surface expression of heteroassociative protein domains (SpyTag/SpyCatcher and SynZip17/18). Programmed aggregation can be used to activate a quorum-sensing circuit, and aggregate size can be tuned via control of the amount of the associative protein displayed on the cell surface. We further demonstrate reversibility of SynZip-mediated assembly by addition of soluble competitor peptide. Genetically programmable bacterial assembly provides a starting point for the development of new applications of engineered microbial communities in environmental technology, agriculture, human health, and bioreactor design.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Aggregates/genetics , Protein Engineering/methods , Escherichia coli Proteins/chemistry , Microorganisms, Genetically-Modified , Peptides/metabolism , Protein Domains/genetics , Quorum Sensing/geneticsABSTRACT
Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as "walking" and "hopping", wherein diffusion is achieved by either partial ("walking") or complete ("hopping") disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.
Subject(s)
Polymers/chemistry , Diffusion , EntropyABSTRACT
Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein-protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein-protein interactions can be used to assemble complex core-shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.
Subject(s)
Bacterial Proteins/metabolism , Colloids/metabolism , Immobilized Proteins/metabolism , Polystyrenes/metabolism , Protein Interaction Maps , Streptococcus pyogenes/metabolism , Bacterial Proteins/chemistry , Colloids/chemistry , Dimerization , Immobilized Proteins/chemistry , Models, Molecular , Particle Size , Polystyrenes/chemistry , Streptococcus pyogenes/chemistryABSTRACT
We present a protein immobilization system, based on the Src Homology 3 (SH3) affinity domain, allowing for a transient interaction between a fibronectin ligand and a biomaterial surface. This strategy leads to enhanced retention of the fibronectin fragment over adsorbed fibronectin, and increased cellular proliferation and motility over either covalently immobilized or adsorbed fibronectin. The results indicate that intermediate affinity protein immobilization could provide benefits for tissue engineering beyond the traditional immobilization techniques, adsorption or covalent attachment.
