Matching Items (17)
Filtering by
- Creators: School of Life Sciences
- Creators: Kuang, Yang
- Creators: Nielsen, David R
- Member of: Theses and Dissertations
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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
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
One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
ContributorsXu, Jimmy (Author) / Nielsen, David (Thesis director) / Wang, Xuan (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering 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