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Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While

Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description

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,

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.

ContributorsBerger, Aubrey (Author) / Pizziconi, Vincent (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor)
Created2022-05
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Description
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications.
ContributorsMorgan, Daylin (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2018