Engineered Living Material Based on Protein-Mediated Bacterial Assembly

Citation

Liu, Hanwei (2024) Engineered Living Material Based on Protein-Mediated Bacterial Assembly. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/6cqr-vq43. https://resolver.caltech.edu/CaltechTHESIS:03182024-051357688

Abstract

Engineered Living Materials(ELMs) is a newly emerging field of biotechnology at the interface of synthetic biology and traditional material science. Over the past few years, several kinds of novel ELMs were developed. These materials, derived from organisms including bacteria, fungi and plants, have potential applications in therapeutics, electronics, constructions and environmental remediation. We invented an novel method that enables bacteria to from cohesive thin films through cell surface display of associative proteins. In this thesis, we will first demonstrate that we can genetically encode the mechanical properties of living bacterial films by controlling amino acid sequences of artificial proteins displayed at cell surface. Later, we will show that we can generate bacterial-matrix composite by displaying enzymes and peptides at the cell surface.

In Chapter 1, we review the development of ELMs and existing examples of ELMs. The fundamental definition of ELMs and trends in ELMs development will be presented. Bacterial based ELMs, created either by encapsulating bacteria of interests into a synthetic polymeric matrix or by boosting the natural biosynthetic pathways of biopolymers and mineralization in bacteria will be the major part of discussion. The goal of this chapter is to provide context and background of ELMs research.

In Chapter 2, we discuss the design and preparation method of our own ELM. The process of how we come up with growing bacterial films on perforated polycarbonate membranes and development of suction coating method will be presented. By using model SpyTag-SpyCatcher bacterial assembly system, we unraveled the principles behind making cohesive bacterial films from a single bacterial colony.

In Chapter 3, we discuss controlling bacterial films’ mechanical properties through genetic manipulation. Engineered bacteria displaying artificial unstructured Elastin-like-peptides (ELPs) at cell surface can form cohesive, soft and yielding films with tens of kPa value of Young’s moduli. By merely adding a cysteine at the N-terminal part of the ELP, the engineered bacteria can form relative tough, non-yielding films with 3 times higher Young’s moduli due to formation of intercellular covalent disulfide bond. Apart from having enhanced mechanical strength, such films containing covalent intercellular interactions have abilities to self-heal within 24 hours after being cut into halves.

In chapter 4, we discuss a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered living bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization.

In chapter 5, we discuss the ability of bacterial protein surface display system to catalyze artificial extracellular matrix formation. We demonstrated that heme-containing peroxidase Apex2 can be fused with autotransporter protein previous described in Chapter 2, 3 and 4, successfully displayed at cell surface and remain functional at catalyzing formation of polymer polyaniline (PANI) in the presence of hydrogen peroxide and aniline monomers at physiological pH. Similarly, by displaying multiple kinds peptide known to mediate silica deposition, we can coat bacteria with silica of different morphologies without reducing the viabilities of bacteria.

In Chapter 6, we discuss the future directions of engineered living materials developed in previous chapters. We propose methods to further improve the mechanical strength of bacterial films, to find “sweet-spot” of biomineralization, and to develop artificial hydrophobin.

Thesis Files