Kendreze Holland

BioE Ph.D. Proposal

October 5th, 2023

12:00 PM

Location: 118 Seminar Room Kendeda 

Committee:

John Blazeck (Ph.D. Advisor)  (School of Chemical & Biomolecular Engineering)

Julie Champion (School of Chemical & Biomolecular Engineering)

Corey Wilson (School of Chemical & Biomolecular Engineering)

Felipe Quiroz (School of Biomedical Engineering, Emory)

William Ratcliff (School of Biological sciences)

A Novel Platform for Simultaneous Control of Multiple Genes at High Throughput

Complex cellular phenotypes and cars are similar in that one can observe their intended purpose (e.g., ability to survive for cells or mobility for cars) but struggle to understand the mechanisms that enable these features. While cars, because they are human-made, can undergo high throughput diagnostics to assess which parts combine to function to allow efficient mobility, methods with an analogous purpose do not exist for complex cellular phenotypes like their ability to survive. Particularly, numerous genes interact in parallel and non-parallel networks to give rise to these complex phenotypes, weakening the understanding gained by testing genes one at a time. In this vein, it is important to note that previous efforts with gene knockout and CRISPR activation/repression studies do not characterize the vast possibilities of achievable gene interactions per cell. Thus, the field of biology needs a high throughput investigative tool with enhanced characterization potential of these intricate gene networks that control complex phenotypes like survival in response to changing environments. To address this shortcoming, we have developed a novel method involving the high throughput creation of multi-single-guide RNA (sgRNA) cassettes. We have shown that it is feasible to assemble multiplex sgRNA cassettes by overlap extension polymerase chain reaction (OE-PCR), and that they can then allow for combinatorial gene expression control in the model organism, Saccharomyces cerevisiae. We will use our novel platform method to simultaneously activate and repress numerous genes to be able to enhance cell survival when exposed to extracellular stressors, such as hydrogen peroxide. Importantly, this technology will have applicability across eukaryotic organisms, providing “research mechanics” with a method that enables improved manipulation of cellular machinery—controlling the expression of multiple genes per cell in a high throughput manner, which is currently an impossible or at least very arduous task.