Ashelyn Sidders, '17

Student

Ashelyn Sidders, '17

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Cohort

2017

Biography

Ashelyn Sidders was born and raised in Orlando, Florida. She is pursuing a bachelor’s degree in both biomedical sciences and biotechnology. Ashelyn is currently conducting research under the guidance of Dr. Kyle Rohde, in order to optimize the current knockout methodology used in pathogenic Mycobacteria to better understand their virulence mechanisms. Additionally, she is also a part of the diagnostics research being done in Dr. Rohde’s lab, and is working to develop a highly sensitive diagnostics system for Multidrug-Resistant Tuberculosis. Ashelyn plans to obtain her Ph.D. in Molecular Pathogenesis and Immunology, to conduct research in the field of emerging infectious diseases.

Faculty Mentor

Dr. Kyle Rohde

Undergraduate Major

Biomedical Sciences/Biotechnology

Future Plans

Ph.D. in Molecular Pathogenesis and Immunology

Research

Detection of Multidrug Resistant Tuberculosis using Loop-Mediated Isothermal Amplification (LAMP) coupled with Binary Deoxyribozyme Sensors

Conducted at the University of Central Florida

Mentors: Dr. Kyle Rohde, University of Central Florida

Abstract: Tuberculosis (TB) affects approximately one third of the world and has been declared a global health emergency by the World Health Organization (WHO). Despite recent advances in the field, drug-resistant TB remains a major problem due to inadequate diagnostic techniques. Early detection of multidrug-resistant TB (MDR-TB) is necessary to ensure correct and timely treatment of resistant strains, thus aiding in eradication of this deadly disease. For this purpose, we have developed an assay, LAMP-DzTfl, utilizing sensitive isothermal amplification of Mtb DNA via loop-mediated isothermal amplification (LAMP) followed by specific detection using binary deoxyribozyme sensors. This assay allows us to rapidly detect the most common mutations responsible for resistance to rifampin (RIF) and isoniazid (INH). Here we demonstrated the use of LAMP-DzTfl, for the specific detection of Mtb and detection of RIF and INH resistance in a clinical isolate. Use of this assay has the potential to enable rapid, and inexpensive diagnosis of drug resistant tuberculosis.

Expanding the genetic toolbox for Mycobacteria: Constructing the pheS counterselection marker

Conducted at the University of Central Florida

Mentors: Dr. Kyle Rohde, University of Central Florida

Abstract: Tuberculosis, recently surpassing HIV/AIDS, is the leading cause of death from a single infectious agent, infecting an estimated 1 in 3 individuals and killing ~1.5 million globally in 2015. Despite the discovery of the causative agent, Mycobacterium tuberculosis (Mtb), over a century ago, scientists are still lacking reliable and efficient tools to investigate genes suspected to be involved in its notable virulence. Mycobacterium abscessus (Mab), an increasingly prevalent cause of death in Cystic Fibrosis patients, lacks usable counterselection methods entirely, as no genetic tools are effective in making Mab gene knockouts (KOs). The goal of this project is to develop a novel counterselection marker utilizing the pheS gene, to further enhance the mycobacterial gene KO strategy used to study uncharacterized genes that may be linked to their virulence. Counterselection, a necessary component of reverse genetics, confers an inability to grow on a selection medium for any bacteria that have not undergone a successful gene KO. In this case, we employ the use of pheS. Encoding for the alpha subunit of phenylalanyl-tRNA synthetase, pheS plays an essential role in microbial physiology by incorporating the amino acid phenylalanine during protein synthesis. Through site-directed mutagenesis we will create a PheS variant (PheS*) that will encode reduced substrate specificity, readily misincorporating the toxic analog, 4CP. The addition of a PheS* counterselection marker into our current suicide vector, pFCKO, will provide optimized selection of desired KOs, as cells that have eliminated the PheS*-containing vector will be able to grow on media containing 4CP.

Summer Research

Committing to Being Different: the Role of PfNFYB in Plasmodium falciparum Gametogenesis

Conducted at the University of California, at San Diego

Mentors: Dr. Elizabeth Winzeler, University of California at San Diego

Abstract: Despite decades of research and global health initiatives to eradicate Malaria, the disease remains a global health crisis with over 200 million new cases, and nearly 450 thousand deaths in 2015. Thus to combat this disease we need rethink the current antimalarial standards in place, as they currently don’t target the transmissible gametocyte stage of Plasmodium falciparum - the causative agent of Malaria – in turn continuing the spread of the disease. To aid in these efforts, our lab is working towards understanding on the genomic level, what causes P. falciparum to enter gametogenesis.

Through directed evolution, we’ve obtained a parasite line lacking the ability to produce gametocytes – the only genomic variation being a single nucleotide variation (SNV) in the gene of a CCAAT-box transcription factor, PfNFYB. This is an intriguing discovery as PfNFYB has been previously found to be downstream of an important stress indicator, cyclic AMP (cAMP), which has previously been shown to affect parasite gametocyte formation. In order to confirm that this SNV in PfNFYB is responsible for the altered phenotype, we’ve engineered additional parasite lines through CRISPR-Cas9 mediated mutagenesis that also illustrate results that parallel the evolved mutant’s lack of gametocyte production. We then performed transcriptome sequencing to identify genes whose transcriptional profile was altered in parasites with the mutated transcription factor. Ultimately, the goal of this project is to understand the functional role of PfNFYB in the induction of gametogenesis between the parental and mutant strains through differential expression of their transcriptomes.

Graduate School

University of North Carolina at Chapel Hill (Ph.D)

Keywords

Biomedical Sciences, Biotechnology

Disciplines

Life Sciences | Microbiology

Ashelyn Sidders, '17

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