Utilizing the Type III Secretion System (T3SS), Shigella spp. uses a cascade of proteins to manipulate, penetrate, and colonize host eukaryotic cells. Inducing epithelial necrosis, Shigella spp. infection is responsible for moderate to severe diarrhea in millions of children and immunocompromised individuals — the majority from under-developed communities. Previous translational research on Shigella spp. has been limited due to the lack of proper in vitro and in vivo models. Understanding infectivity of Shigella spp. heavily relies on imprecise estimations of intracellular Shigella spp., which ultimately impacts vaccine and antibiotic efforts. This project aims to address this problem by developing a novel red intracellular reporter to quantify successful invasion of Shigella flexneri. By using a series of polymerase chain reaction (PCR) assemblies, we aim to construct a plasmid with a RFP reporter to be expressed during successful invasion of Shigella spp. The IpaH9.8 MxiE promoter, which has been shown to be expressed upon cell entry, has been integrated within the pUltra RFP plasmid through a Gibson-Reaction assembly and cloned using PCR. The new DNA replicate was electroporated into a streptomycin-resistant S. flexneri strain. We  initially tested each strain in vitro by inoculating HCT-8 cells with the newly engineered S. flexneri and monitored for selective RFP expression by intracellular S. flexneri. The outcomes of this project will provide an accurate and efficient method of quantifying invasive S. flexneri. in vitro and in vivo, as well as quantifying efficacy of new antibiotic treatments. The implications of this project are crucial to the advancement of shigellosis research and in furthering the efforts of the international community to abate the rates of disease mortality and burden.

Characterizing biological functions on a single molecule scale increases understanding of biological functions by providing information about the indiviual contributions which combine to create larger scale functions. Single molecule measurements are a crucial part of characterizing molecular interactions. Although atomic force microscopy (AFM) and magnetic tweezers are able to measure the response of single molecules to mechanical force, it is challenging to ensure single molecules are being measured. In this project, a precise DNA Origami structure was used to space molecules for single molecule force measurements. Base pair association between DNA nucleotides allowed specific nanostructures to be designed and fabricated. Molecules of interest self-assemble to specific sites of the structure. AFM was used for imaging and obtaining force measurements. This research investigates the strength of adhesion for double stranded DNA when subjected to different loading rates as a proof of concept. In the future, this structure will be used to determine force properties of diverse molecular interactions like platelet and bacterial adhesions.

DNA origami nanotechnology has evolved rapidly since its conception eleven years ago. Both two-dimensional and three-dimensional nanostructures have been created with potential applications in targeted drug delivery, “smart” diagnostic technology, and the study of cell behavior. By annealing “staple” oligonucleotide strands to a single-stranded DNA scaffold we can effectively fold the DNA onto itself to build the nanostructures of interest. One of the primary physical limitations to what one can build is the scaffold. The most commonly used scaffold is derived from the bacteriophage M13mp18 and has a length of 7,249 nucleotides. Its length has previously been varied; however, an overlooked limitation is the secondary structure DNA naturally exhibits. These are sites in which the scaffold binds to itself, thus creating competition for staples to bind during folding reactions. To predict the impact that a designed sequence with little secondary structure could have, we analyzed the first 6,000 bases of the M13mp18 DNA sequence using NUPACK, a nucleic acid sequence analyzer, for their minimum free energy (MFE) at storage, manipulation, and maximum folding reaction temperature. Preliminary data shows M13mp18 exhibits less secondary structure at a high temperature (65°C) than at a low temperature (4°C) and increasing the concentration of divalent salts linearly increases the amount of secondary structure. Additionally, alternative, shorter sequences have been engineered and their secondary structure is being analyzed at varying conditions. To further determine the effects on yield and stability, structures will be folded using the designed sequence and the standard sequence as a scaffold. These will be compared through agarose gel electrophoresis and transmission electron microscopy. The results from this preliminary data could help us move us toward using a scaffold with decreased secondary structure present at folding temperatures which could potentially result in higher yields, shorter folding reactions, and increased stability.

Energetic tradeoffs are responsible for the shift of energy allocation between various physiological processes within an organism which define life history traits. By understanding energetic tradeoffs, we can make predictions about growth and maturation during different life history stages. In aquacultured species, such life history traits determine meat production and time to harvest, and are thus important for profitability of operations. The purple-hinged rock scallop is being developed for commercial production in Puget Sound. Two common products for scallop meat are adductor muscle and the whole animal served on the half shell. Understanding the relationship between maturity of the gonad and size of the adductor muscle would inform market choice between these options for the emerging industry. Previous research on scallop energy allocation suggests that scallops may allocate energy away from the adductor and into the gonad during maturation. To evaluate this energetic tradeoff in rock scallops, 1200 scallops were out-planted in Puget Sound, 400 at each of 3 sites: Neah Bay, Dabob Bay, and South Puget Sound. Shell height, shell width, and shell depth were measured at three time points: initial outplanting (10/28/16), a sampling midpoint (03/06/17), and final sampling (06/08/17 – 06/11/17). Final sampling also included whole weight, meat weight, adductor diameter, and samples of gonad tissue for histology. The gonad tissue samples were mounted on slides, and analyzed to determine sexual maturation levels. Using maturation ratios, I will investigate whether there is correlation between adductor size and maturation, and whether any detected correlation is consistent across outplanting sites. Such a correlation would provide evidence of an energetic tradeoff during maturation as an individual shifts energy stored in the adductor muscle into the gonad. This data will build on previous research and inform the emerging rock scallop aquaculture industry on market selection.