Protein-Mediated Colloidal Assembly

Citation

Silverman, Bradley Ross (2020) Protein-Mediated Colloidal Assembly. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x3ya-fq67. https://resolver.caltech.edu/CaltechTHESIS:05302020-111741817

Abstract

The assembly of colloidal-sized particles into larger structures by the manipulation of inter-particle forces has been a subject of significant research towards applications in materials science, soft matter physics, and synthetic biology. To date, much of this work has utilized manipulation of electrostatic or depletion interactions to drive the aggregation of the particles. More recently, specific (bio)-chemical interactions have been harnessed, particularly the use of deoxyribonucleic acid (DNA) linkers to program particle interactions by Watson-Crick base-pairing. In this thesis, we will demonstrate the use of an alternative set of biochemical interactions, protein-protein interactions, which have useful properties (in particular, their ability to be completely genetically-programmable).

In Chapter 2, we discuss the development of a model system for the protein-mediated assembly of colloidal micro-particles. Associative proteins are grafted onto the surface of polystyrene micro-particles, enabling their assembly into aggregates either through reversible coiled-coil interactions or by irreversible isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of the immobilized associative proteins on their surface. Further, we show that particles grafted with different protein pairs show excellent self-sorting into separate aggregates. Finally, we demonstrate that these protein-protein interactions can be used to assemble complex core-shell aggregates. The principles of protein-mediated colloidal assembly learned in this chapter will be instructive as we attempt the more complex assembly of living microbial cells.

In Chapter 3, we discuss the implementation of a protein-driven aggregation system in living bacterial cells. Similarly to Chapter 2, we demonstrate that we can drive the aggregation of bacteria by the surface display of proteins enabling reversible coiled-coil interactions or irreversible isopeptide bonds. The sizes of these aggregates are tunable by titration of surface expression levels by standard synthetic biology techniques. Finally, we show that this programmable aggregation of bacteria may have physiological consequences for the cells, in particular, the activation of a quorum sensing circuit due to a higher local concentration of bacteria.

In Chapter 4, we further investigate how the properties of the aggregates described in Chapter 3 can be controlled and how these relate to the underlying properties of the associative proteins and shear field. we demonstrate control of the assembly kinetics and equilibrium sizes of the resulting flocs over several orders of magnitude using different associating proteins and expression levels. Finally, we show that a single point mutation in the associative protein leads to an unexpected ultra-sensitive pH-responsive coil, demonstrating the importance of molecular-scale interactions on the macro-scale properties of the aggregates.

In Chapter 5, we discuss the ability of the bacterial aggregates described in Chapters 3 and 4 to enable substrate channeling between bacterial strains, leading to enhancement of titers in multi-step biosynthetic pathways. When biosynthetic pathways are split into separate bacterial strains, dilution of the intermediate compound into the bulk media may decrease reaction flux. By aggregating the bacteria, the intermediate compound is able to rapidly diffuse into the downstream cell without being diluted, enabling higher reaction fluxes. we demonstrate through the model flavonoid synthesis pathway that aggregation can lead to substantially higher titers of the desired compound without pathway re-engineering, and develop a mathematical model by which this result can be understood.

Thesis Files