Probing the Spt16-Pob3 Interface Through BPA Cross-Linking in Saccharomyces cerevisiae as a Proof of Concept

Examining protein-protein interactions in vivo can often be challenging due to nonspecific cross reactivity. The purpose of this study is to determine the effectiveness of the BPA cross-linking technique in Saccharomyces cerevisiae by confirming protein-protein interactions between FACT subunits: Spt16 and Pob3. We concluded that the BPA cross-linking technique was effective in confirming the protein-protein interactions between FACT subunits within the dimerization domain

Using Pre-Cleaned Coverslips to Optimize Coverslip Preparation for Single-Molecule Fluorescence Microscopy

Single-molecule fluorescence microscopy has become a popular tool for exploring structural changes and dynamics of biological systems. In our laboratory, we use single-molecule techniques to track conformational changes of immobilized nitric oxide synthase (NOS) and determine their relationship to catalytic activity. Properly immobilizing biomolecules like NOS on a glass surface requires careful attention to coverslip cleaning and preparation. There are many protocols available for cleaning glass coverslips, but these protocols are time-consuming and often use harsh conditions. Alternatively, commercially cleaned and passivated coverslips are available but are quite expensive. In this poster, we examine the possibility of using purchased pre-cleaned coverslips (Schott Nexterion) that come ready to be prepared for single-molecule measurements. We present figures and measurements of merit comparing the pre-cleaned coverslips with ozone cleaned coverslips; demonstrating the effectiveness of pre-cleaned coverslips for single-molecule fluorescence microscopy

Investigating Gene Functions in Mycobacteriophage Island3

Island3 is a temperate Cluster I1 mycobacteriophage that infects Mycobacterium smegmatis mc2155. Its genome consists of 76 protein-coding genes, only 17 of which have known functions. Towards the goal of identifying additional gene functions, we amplified, cloned, and assayed 14 genes for host cytotoxicity and the ability to render the host resistant to infection by other phages (defense). We analyzed genes 10, 11, 12, 13, 14, 15, 21, 22, 25, 50, 51, 57, 60, and 61 and concluded that none of these genes exhibited either host cytotoxicity or defense against phage infection. We are in the process of assaying the remaining genes of Island3

Exploring a Transcriptional Regulatory Region in Mycobacteriophage JacoRen57

Mycobacteriophages are infectious particles that infect mycobacteria, and little is known about cis-regulatory elements that control their gene expression. In phage genomes, cis-regulatory elements commonly precede a series of genes that are expressed as an operon. JacoRen57 is a cluster AB mycobacteriophage that possesses forward and reverse genes with non-coding gaps interspersed throughout its genome. We assayed one of the gap regions of JacoRen57 (40644-40974 bps) for regulatory activity in the downstream direction when present in its host, Mycobacterium smegmatis, by cloning the region into pLO86, a vector containing the mCherry reporter gene. The putative regulatory region induced the expression of mCherry in vivo, indicating the presence of a promoter in this region of the JacoRen57 genome. Utilizing 5’ deletions analysis, we identified promoter and repressor elements within this regulatory region. We are conducting further experiments to understand the characteristics of the repressor region and which sigma factor/s binds to this promoter

Exploring a Putative Promoter Region in Mycobacteriophage JacoRen57

Phages are abundant particles that infect bacteria. For the SEA-PHAGES program, students discover phages and annotate their genomes. Throughout the annotation process, genes are identified based on bioinformatics evidence; however, little is known about mycobacteriophage promoters as they are not annotated. Promoters are necessary for gene expression, and in mycobacteriophages, a promoter typically precedes a series of genes that are expressed as a single transcript from which multiple proteins are translated. JacoRen57 is a singleton mycobacteriophage with a siphoviridae morphotype that possesses forward and reverse genes with gaps located at the transitions from forward to reverse genes. We hypothesized that these gaps contain promoters. We used BPROM and PePPER, prokaryotic promoter predictor software, which yielded matches to promoter consensus sequences in one of the gap regions. We cloned the putative promoter region into pLO86, a vector containing the mCherry reporter gene, to determine if the cloned region functions as a promoter by inducing mCherryexpression in Mycobacterium smegmatis. The putative promoter region did not function as a promoter in vivo under standard M. smegmatisgrowth conditions

Annotation of Two Soil Mycobacteriophages: JacoRen57 and DrLupo

We discovered two novel Mycobacteriophagesfrom soil samples. JacoRen57 is a singleton, most closely related to cluster AB phages. DrLupois a member of the rare H2 sub-cluster containing only one other member. Both phages exhibit a siphoviridaemorphotype, double-stranded DNA genome, and a non-contractile tail. The genome of JacoRen57 is composed of 52,411 base pairs with a 56.7% GC content and includes genes and plaque morphology that suggest a lytic life cycle. The genome is organized according to the following order from the left to right arm: 33 forward genes followed by 16 reverse genes and 24 forward genes. We identified functions for 33 of the 73 protein coding genes using bioinformatics software. The 70,030 base-pairgenome of DrLupohas a 57.5% GC content and contains genes that support a lytic life cycle. We identified 110 forward genes and assigned functions to 25 of them

Elucidating Antiproliferative Mechanisms of Grapeseed, Guava, and Juniper Berry Extracts

Plant extracts are an untapped source of medicinal potential. Even today they are used as standalone treatments and applied alongside conventional therapies. The focus of our laboratory is to identify plant extracts exhibiting antiproliferative activity in vitro, to determine which chemicals are responsible for this activity, and to elucidate mechanism(s) by which growth is slowed/inhibited by plant extracts. Specifically, we exposed five cell lines/strains to twenty-two plant extracts and measured cell proliferation. Extracts from Vinca, Juniper Berry, Guava, Grapeseed, and Yew slowed the growth of all five lines/strains in a dose dependent fashion. We are working to understand the mechanism of antiproliferation by measuring induction of apoptosis, effects on microtubule assembly, and wound healing

Stormbreaker8 and A3Wally Bacteriophage Genome Annotations

Stormbreaker8 and A3Wally are two novel bacteriophages isolated and purified on Microbacterium foliorum NRRL B-24224 by students in the Fall 2020 Discovery course. Stormbreaker8, an EA1 cluster lytic phage, was isolated from soil collected in Orange City, IA. Its circular permuted genome contains 41,751 base-pairs with 63.4% GC content. A3Wally, a GD cluster phage, was isolated from soil collected in Sioux Center, IA. Its genome is 60.1% GC, contains 194,724 base-pairs, and its ends are direct terminal repeats. Spring 2021 Genetics students annotated the genomes using bioinformatics software

Adaptive Market Hypothesis: Evidence from three centuries of UK data

We examine the evolving efficiency of UK stock market and currency (British Pound) during the last three centuries. Using both Automatic Variance Ratio (AVR) and Automatic Portmanteau (AQ) tests, we find evidence of time-varying degree of efficiency which supports the Adaptive Markets Hypothesis (AMH)