(7al) Exploiting Organization in Bacteria for Synthetic Biology

Bacteria were once thought to be simple bags of enzymes. Today, we understand that bacteria have levels of organization that are more complex than previously thought. My research interest is in exploiting bacterial organization for useful synthetic biology applications. This includes exploring how spatial organization, temporal organization, and structural organization enhance the efficiency of cellular processes. Bacterial microcompartments are an excellent example of how bacteria utilize spatial and temporal organization. Approximately 200 nm in diameter, these structures are composed of thousands of subunits of hexameric shell proteins that encapsulate the necessary enzymes to carry out specific metabolic pathways. These pathways often produce toxic intermediates which are sequestered within the compartment, thereby protecting the rest of the cell. Using a flow-cytometry based assay for quantifying the amount of protein encapsulation, I have elucidated the design rules for an N-terminal encapsulation sequence, and have shown that a variety of heterologous proteins can be encapsulated at titratable quantities within bacterial microcompartments using variations of a short amino acid tag.

Bacterial endospores provide an example of complex structural organization. In response to nutrient starvation conditions, certain bacteria such as Bacillus subtilis can elaborate a layered protein coat comprising of approximately 80 different proteins, protecting the dormant endospore from chemical attacks, dessication, and radiation. I have worked to understand how the very first coat protein, SpoVM, localizes specifically to the developing forespore membrane within the mother cell by recognizing its convex membrane curvature. Using solid-supported lipid bilayers on silica microbeads, we reconstituted bacterial membranes in vitro and characterized the binding of SpoVM to membranes with different radii of curvature. We found that the mechanism behind the preferential binding to membranes with high degrees of curvature results from a higher kinetic adsorption rate coupled with binding cooperativity, and that this property is critically dependent on a structural kink in the protein structure that delineates the 26-amino acid peptide into an unstructured flexible N-terminus and an amphipathic alpha helical C-terminus.

Looking ahead, the groundwork I established in elucidating design principles and mechanistic understanding of bacterial organizational systems will enable my future lab to take these natural systems and exploit them for synthetic biology applications. I propose to utilize the organization of microcompartment to create customized microenvironments within the lumen that allows for synthetic pathways that are generally incompatible within the bulk cytosol. These self-contained “nanoreactors” within the bacterium can be tailored to be, for example, anaerobic, and can greatly expand the repertoire of pathways capable of being expressed in bacteria. I also propose to exploit the structural organization of bacterial endospores to create small molecule delivery systems that utilize the structural and chemical stability of the endospore coat proteins. Such a system can shield the cargo within from chemical and enzymatic attacks and increase its half-life in vivo, and also provides a platform for decorating the particle surface with additional proteins or biomolecules. Together, these proposed works to exploit the organization in bacteria will enhance the suite of biological tools available to synthetic biologists.

Teaching Interests:

My dedication to education is a major component in my decision to pursue a career in academia. I have served as a graduate student instructor for two chemical engineering courses: transport and separations, a core class taken by all undergraduates in the chemical engineering program, and biochemical engineering, an elective taken by chemical engineering majors that seek a concentration in biotechnology. In these courses, I used various strategies to actively engage students in learning, which included learning from peers, and supplementing lectures with activities designed to engage students with different learning styles. I have found great success with these strategies, as measured by positive course evaluation comments and ratings, as well as two teaching awards – the Dow Excellence in Teaching Award, awarded with a cash prize, and the campus-wide Outstanding Graduate Student Instructor award. As an instructor, I plan to keep up to date with pedagogical methods, and adapt my teaching style to fit the needs of my students, especially as new methods of learning are continually emerging.