Matching Items (17)
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- All Subjects: Synthetic Biology
- Creators: Wang, Xiao
- Member of: Theses and Dissertations
- Member of: ASU Electronic Theses and Dissertations
Description
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
Description
The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here we employ engineered variants of CRISPR systems in proof-of-principle experiments demonstrating the ability of CRISPR-Cas derived single-DNA-strand cutting enzymes (nickases) to direct host-cell genomic recombination. E.coli is generally regarded as a poorly recombinogenic host with double-stranded DNA breaks being highly lethal. However, CRISPR-guided nickase systems can be easily programmed to make very precise, non-lethal, incisions in genomic regions directing both single reporter gene and larger-scale recombination events deleting up to 36 genes. Genome integrated repetitive elements of variable sizes can be employed as sites for CRISPR induced recombination. We project that single-stranded based editing methodologies can be employed alongside preexisting genome engineering techniques to assist and expedite metabolic engineering and minimalized genome research.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis director) / Haynes, Karmella (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR based transcriptional regulators have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. However, for this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Here, we discuss recent advances and integrative strategies that can help pave the way towards a new class of transcriptional therapeutics.
ContributorsPandelakis, Matthew (Author) / Ebrahimkhani, Mohammad (Thesis director) / Kiani, Samira (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic range genetic regulation are necessary. Currently the field struggles to demonstrate reliable regulators which are programmable and specific, yet also allow for a high dynamic range of control. Inspired by the characteristics of the RNA toehold switch in E. coli, this project attempts utilize artificial introns and complementary trans-acting RNAs for gene regulation in a eukaryote host, S. cerevisiae. Following modification to an artificial intron, splicing control with RNA hairpins was demonstrated. Temperature shifts led to increased protein production likely due to increased splicing due to hairpin loosening. Progress is underway to demonstrate trans-acting RNA interaction to control splicing. With continued development, we hope to provide a programmable, specific, and effective means for translational gene regulation in S. cerevisae.
ContributorsDorr, Brandon Arthur (Author) / Wang, Xiao (Thesis director) / Green, Alexander (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.
ContributorsBarclay, David (Co-author) / Simper, Jan (Co-author) / Haynes, Karmella (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Over the past 20 years, the fields of synthetic biology and synthetic biosystems engineering have grown into mature disciplines, leading to significant breakthroughs in cancer research, diagnostics, cell-based medicines, biochemical production, etc. Application of mathematical modelling to biological and biochemical systems have not only given great insight into how these systems function, but also have lent enough predictive power to aid in the forward-engineering of synthetic constructs. However, progress has been impeded by several modes of context-dependence unique to biological and biochemical systems that are not seen in traditional engineering disciplines, resulting in the need for lengthy design-build-test cycles before functional prototypes are generated.In this work, two of these universal modes of context dependence – resource competition and growth feedback –their effects on synthetic gene circuits and potential control mechanisms, are studied and characterized. Results demonstrate that a novel competitive control architecture can be utilized to mitigate the effects of winner-take-all resource competition (a form of context dependence where distinct gene modules influence each other by competing over a shared pool of transcriptional/translational resources) in synthetic gene circuits and restore circuits to their intended function. Application of the fluctuation-dissipation theorem and rigorous stochastic simulations demonstrate that realistic resource constraints present in cells at the transcriptional and translational levels influence noise in gene circuits in a nonmonotonic fashion, either increasing or decreasing noise depending on the transcriptional/translational capacity.
Growth feedback on the other hand links circuit function to cellular growth rate via increased protein dilution rate during exponential growth phase. This in turn can result in the collapse of bistable gene circuits as the accelerated dilution rate forces switches in a high stable state to fall to a low stable state. Mathematical modelling and experimental data demonstrate that application of repressive links can insulate sensitive parts of gene circuits against growth-fluctuations and can in turn increase the robustness of multistable circuits in growth contexts.
The results presented in this work aid in the accumulation of understanding of biological and biochemical context dependence, and corresponding control strategies and design principles engineers can utilize to mitigate these effects.
ContributorsStone, Austin (Author) / Tian, Xiao-jun (Thesis advisor) / Wang, Xiao (Committee member) / Smith, Barbara (Committee member) / Kuang, Yang (Committee member) / Cheng, Albert (Committee member) / Arizona State University (Publisher)
Created2023
Description
Ecology has been an actively studied topic recently, along with the rapid development of human microbiota-based technology. Scientists have made remarkable progress using bioinformatics tools to identify species and analyze composition. However, a thorough understanding of interspecies interactions of microbial ecosystems is still lacking, which has been a significant obstacle in the further development of related technologies. In this work, a genetic circuit design principle with synthetic biology approaches is developed to form two-strain microbial consortia with different inter-strain interactions. The microbial systems are well-defined and inducible. Co-culture experiment results show that our microbial consortia behave consistently with previous ecological knowledge and thus serves as excellent model systems to simulate ecosystems with similar interactions. Colony patterns also emerge when co-culturing multiple species on solid media. With the engineered microbial consortia, image-processing based methods were developed to quantify the shape of co-culture colonies and distinguish microbial consortia with different interactions. Factors that affect the population ratios were identified through induction and variations in the inoculation process. Further time-lapse experiments revealed the basic rules of colony growth, composition variation, patterning, and how spatial factors impact the co-culture colony.
ContributorsChen, Xingwen (Author) / Wang, Xiao (Thesis advisor) / Kuang, Yang (Committee member) / Tian, Xiaojun (Committee member) / Brafman, David (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2022
Description
The mutual inhibition between synthetic gene circuits and cell growth produces growth feedback in the host-circuit system. Previous studies have demonstrated that the growth feedback has an marked impact on the molecular dynamics of the host-circuit system. However, the complexity of the growth feedback effect is not fully understood. A theoretical framework was developed to study the dynamics of the coupling between growth feedback and synthetic gene circuits. The study’s results reveal three major points about the impact of growth feedback. First, a nonlinear emergent behavior mediated by growth feedback. The unexpected behavior depends on the dynamic ribosome allocation between gene circuit expression and host cell growth. Second, the emergence and loss of unexpected qualitative states on the host-circuit system generated by ultrasensitive growth feedback. Third, the growth feedback-induced cooperativity behavior in synthetic gene modules competing for resources. In addition, growth feedback attenuated the winner-takes-all rules on resource competition between the two self-activating modules. These results demonstrate that growth feedback plays an important role in the host-circuit system’s molecular dynamics. Characterizing general principles from the effect of growth facilitates the ability to minimize or even harness unexpected gene expression behaviors derived from the effect of growth feedback.
ContributorsMelendez-Alvarez, Juan Ramon (Author) / Tian, Xiaojun (Thesis advisor) / Wang, Xiao (Committee member) / Kuang, Yang (Committee member) / Arizona State University (Publisher)
Created2022
Description
Synthetic biology (SB) has become an important field of science focusing on designing and engineering new biological parts and systems, or re-designing existing biological systems for useful purposes. The dramatic growth of SB throughout the past two decades has not only provided us numerous achievements, but also brought us more timely and underexplored problems. In SB's entire history, mathematical modeling has always been an indispensable approach to predict the experimental outcomes, improve experimental design and obtain mechanism-understanding of the biological systems. \textit{Escherichia coli} (\textit{E. coli}) is one of the most important experimental platforms, its growth dynamics is the major research objective in this dissertation. Chapter 2 employs a reaction-diffusion model to predict the \textit{E. coli} colony growth on a semi-solid agar plate under multiple controls. In that chapter, a density-dependent diffusion model with non-monotonic growth to capture the colony's non-linear growth profile is introduced. Findings of the new model to experimental data are compared and contrasted with those from other proposed models. In addition, the cross-sectional profile of the colony are computed and compared with experimental data. \textit{E. coli} colony is also used to perform spatial patterns driven by designed gene circuits. In Chapter 3, a gene circuit (MINPAC) and its corresponding pattern formation results are presented. Specifically, a series of partial differential equation (PDE) models are developed to describe the pattern formation driven by the MINPAC circuit. Model simulations of the patterns based on different experimental conditions and numerical analysis of the models to obtain a deeper understanding of the mechanisms are performed and discussed. Mathematical analysis of the simplified models, including traveling wave analysis and local stability analysis, is also presented and used to explore the control strategies of the pattern formation. The interaction between the gene circuit and the host \textit{E. coli} may be crucial and even greatly affect the experimental outcomes. Chapter 4 focuses on the growth feedback between the circuit and the host cell under different nutrient conditions. Two ordinary differential equation (ODE) models are developed to describe such feedback with nutrient variation. Preliminary results on data fitting using both two models and the model dynamical analysis are included.
ContributorsHe, Changhan (Author) / Kuang, Yang (Thesis advisor) / Wang, Xiao (Committee member) / Kostelich, Eric (Committee member) / Tian, Xiaojun (Committee member) / Gumel, Abba (Committee member) / Arizona State University (Publisher)
Created2021