Analytical Sensors, Surface Chemistry, Array Fabrication, 3D Cell Culture Systems, Molecular Recognition, Protein-DNA interactions, Synthetic Biology
University of Pittsburgh, BS in Chemistry (2005); University of Wisconsin-Madison, PhD in Chemistry with Lloyd M. Smith (2009); Harvard University, Postdoctoral Fellow with George M. Whitesides (2010-2013)
Chemical communication â€”the interaction of two or more chemical components that alter the flow of information within a biological systemâ€” ranges in complexity from the binding of a single inhibitor to an enzyme to a multi-step signaling pathway. Research in the Lockett group uses a multi-disciplinary approachâ€”combining aspects of chemistry, materials science, biochemistry, and molecular biologyâ€”to develop new analytical tools and in vitro assays that can predict what interactions are occurring in a cell or in a community of cells. We are particularly interested in surface-based assays that closely mimic in vivo conditions, are high-throughput in nature, are easily assembled and performed, and provide quantitative data.
It is currently not clear if the binding of proteins to a double-stranded DNA (dsDNA) molecule attached to a solid support is representative of binding in solution, or, more importantly, in vivo. The promise of â€œdesigner transcription factorsâ€ in which the active site of a protein is engineered to modulate its binding affinity to particular sequence of DNA is currently limited by a lack of methods to screen for the affinity and selectivity of DNA-protein interactions.
We are developing new surface chemistries to prepare arrays of dsDNA molecules on planar, carbon-based substrates to screen protein-DNA interactions. The goal of this project is an array in which we can quantify protein-DNA interactions on a genome-wide level, and obtain binding information that correctly reflects protein-DNA interactions in vivo.
The Metabolism of Xenobiotics
Cytochrome P450s are membrane-bound proteins that are responsible for metabolizing, or in some instances, activating, xenobiotics in the human body. There are a number of in vitro assays to screen xenobiotic metabolism that utilize variants of CYP450 enzymes that do not contain the membrane binding domain. It is currently not clear if the activity of these truncated enzymes is representative of full-length, membrane-bound CYP450s.
We are also developing surface chemistries to prepare arrays of membrane proteins. The goal of this project is an array in which we can screen and quantify the metabolism of a xenobiotic in the presence of a single CYP450 enzyme, or multiple CYP450s working in concert.
The Phenotypic Response of Cells in Metabolically Stressed Environments
Communities of cellsâ€”whether they are the numerous species of bacteria that compose a biofilm, or the different cell types that compose human tissue â€”communicate through the formation of gradients of signaling molecules.
We are developing three-dimensional constructs to culture cells. There is a general consensus that the phenotype and behavior of cells is markedly different in two- and three-dimensional cultures, and we believe an assay that mimics the environment of mammalian cells in a tissue or bacterial cells in a biofilm will provide a more accurate picture of what factors cause cells to become mobile, for example a tumorigenic cell, or dormant, for example a bacterial persister cell.