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- Creators: Wang, Xiao
- Creators: Kiani, Samira
- Creators: Muller, Ryan
- Status: Published
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
Regenerative medicine utilizes living cells as therapeutics to replace or repair damaged or diseased tissue, but the manufacturing processes to produce cell-based tissue products require customized biounit operations that do not currently exist as conventional biochemical and biopharma manufacturing processes. Living cells are constantly changing and reacting to their environment, which in the case of cells isolated from their hosts, are utilized as living bioreactor components that, by themselves, are manipulated to biomanufacturer selected tissue products. Therefore, specialized technology is required to assure that cellular products produce the phenotypical tissue characteristics that the final product is designated to have, while also maintaining sterility of the culture. Because of this, FDA guidelines encourage the use of Process Analytical Technology (PAT – see Ref ) to be integrated into manufacturing systems of biologics to ensure quality and safety. To address the need for evaluation of sensor technologies for potential use in PAT, a literature review of both existing sensing technologies and biomarkers was conducted. After a thorough assessment of the sensor technologies that were most applicable to biomanufacturing, spectrophotometry was selected to monitor the metabolic components glucose and lactate of living cells in culture in real time. Initially, spectrophotometric measurements were taken of mock solutions of glucose and lactate solutions at concentrations relevant to human cell culture and physiology. With that data, a mathematical model was developed to predict a solution’s glucose and lactate concentration. This model was then integrated into a Matlab program that was used to continuously monitor and estimate solutions of glucose and lactate concentrations in real time. After testing the accuracy of this program in different solutions, it was determined that calibration curves and models must be made for each media type and estimates of glucose and lactate were found accurate only at higher concentrations. This program was successfully utilized to monitor in real time glucose and lactate production and consumption trends of Mesenchymal Stem Cells (MSCs) in culture, demonstrating proof-of-concept of the proposed bioprocess monitoring schema.