Progress toward a cloneable bismuth nanoparticle

Abstract

Electron microscopy (EM) is a technique that obtains high resolution biological images. However, a major limitation is inadequate contrast between a protein of interest and the surrounding cellular background. To overcome this challenge, the Ackerson lab has developed a cloneable nanoparticles (cNPs) which is described as an inorganic particle enzymatic synthesized using a metal reducing enzymes. These electron dense nanoparticles serve as a contrast marker, enhancing the contrast between a protein of interest and the cellular background. The focus of my thesis is to create a small compact bismuth cNPs to improve contrast labelling in EM. The first chapter provides an overview of current biological imaging and introduces how cloneable nanoparticles have promising application as a contrast marker. It reviews the role of essential intracellular metals and details the development of a cloneable selenium nanoparticle made from a metal reducing enzyme, Glutathione Reductase-Like Metalloid Reductase (GRLMR), which reduces selenite to form zero valance selenium. This chapter concludes by outlining the advantages of using bismuth as a contrasting marker. The second chapter discusses the background of arsenate reductase (ArsC) which natively reduces arsenate to arsenite. Given the chemical and structural properties as arsenite and bismuth, I hypothesized that ArsC may be capable of reducing bismuth(III) glutathione. This hypothesis was tested through enzymatic activity assays and by observing particle formation using analytical techniques to determine whether ArsC can reduce Bi(GSH)3. The third chapter explores how a directed evolution approach is used to enhance ArsC's selectivity towards reducing Bi(GSH)3. I generated a random mutagenesis library of ArsC variants, with the next step focusing on screening or selecting for variants that exhibit improved activity towards Bi(GSH)3.