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- All Subjects: Biomedical Engineering
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
The development of biosensing platforms not only has an immediate lifesaving effect but also has a significant socio-economic impact. In this dissertation, three very important biomarkers with immense importance were chosen for further investigation, reducing the technological gap and improving their sensing platform.Firstly, gold nanoparticles (AuNP) aggregation and sedimentation-based assays were developed for the sensitive, specific, and rapid detection of Ebola virus secreted glycoprotein (sGP)and severe acute respiratory syndrome coronavirus 2 (SARS-COV2) receptor-binding domain (RBD) antigens. An extensive study was done to develop a complete assay workflow from critical nanobody generation to optimization of AuNP size for rapid detection. A rapid portable electronic reader costing (<$5, <100 cm3), and digital data output was developed. Together with the developed workflow, this portable electronic reader showed a high sensitivity (limit of detection of ~10 pg/mL, or 0.13 pM for sGP and ~40 pg/mL, or ~1.3 pM for RBD in diluted human serum), a high specificity, a large dynamic range (~7 logs), and accelerated readout within minutes. Secondly, A general framework was established for small molecule detection using plasmonic metal nanoparticles through wide-ranging investigation and optimization of assay parameters with demonstrated detection of Cannabidiol (CBD). An unfiltered assay suitable for personalized dosage monitoring was developed and demonstrated. A portable electronic reader demonstrated optoelectronic detection of CBD with a limit of detection (LOD) of <100 pM in urine and saliva, a large dynamic range (5 logs), and a high specificity that differentiates closely related Tetrahydrocannabinol (THC). Finally, with careful biomolecular design and expansion of the portable reader to a dual-wavelength detector the classification of antibodies based on their affinity to SARS-COV2 RBD and their ability to neutralize the RBD from binding to the human Angiotensin-Converting Enzyme 2 (ACE2) was demonstrated with the capability to detect antibody concentration as low as 1 pM and observed neutralization starting as low as 10 pM with different viral load and variant. This portable, low-cost, and versatile readout system holds great promise for rapid, digital, and portable data collection in the field of biosensing.
ContributorsIkbal, Md Ashif (Author) / Wang, Chao (Thesis advisor) / Goryll, Michael (Committee member) / Zhao, Yuji (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2022
Description
Ideas from coding theory are employed to theoretically demonstrate the engineering of mutation-tolerant genes, genes that can sustain up to some arbitrarily chosen number of mutations and still express the originally intended protein. Attention is restricted to tolerating substitution mutations. Future advances in genomic engineering will make possible the ability to synthesize entire genomes from scratch. This presents an opportunity to embed desirable capabilities like mutation-tolerance, which will be useful in preventing cell deaths in organisms intended for research or industrial applications in highly mutagenic environments. In the extreme case, mutation-tolerant genes (mutols) can make organisms resistant to retroviral infections.
An algebraic representation of the nucleotide bases is developed. This algebraic representation makes it possible to convert nucleotide sequences into algebraic sequences, apply mathematical ideas and convert results back into nucleotide terms. Using the algebra developed, a mapping is found from the naturally-occurring codons to an alternative set of codons which makes genes constructed from them mutation-tolerant, provided no more than one substitution mutation occurs per codon. The ideas discussed naturally extend to finding codons that can tolerate t arbitrarily chosen number of mutations per codon. Finally, random substitution events are simulated in both a wild-type green fluorescent protein (GFP) gene and its mutol variant and the amino acid sequence expressed from each post-mutation is compared with the amino acid sequence pre-mutation.
This work assumes the existence of synthetic protein-assembling entities that function like tRNAs but can read k nucleotides at a time, with k greater than or equal to 5. The realization of this assumption is presented as a challenge to the research community.
An algebraic representation of the nucleotide bases is developed. This algebraic representation makes it possible to convert nucleotide sequences into algebraic sequences, apply mathematical ideas and convert results back into nucleotide terms. Using the algebra developed, a mapping is found from the naturally-occurring codons to an alternative set of codons which makes genes constructed from them mutation-tolerant, provided no more than one substitution mutation occurs per codon. The ideas discussed naturally extend to finding codons that can tolerate t arbitrarily chosen number of mutations per codon. Finally, random substitution events are simulated in both a wild-type green fluorescent protein (GFP) gene and its mutol variant and the amino acid sequence expressed from each post-mutation is compared with the amino acid sequence pre-mutation.
This work assumes the existence of synthetic protein-assembling entities that function like tRNAs but can read k nucleotides at a time, with k greater than or equal to 5. The realization of this assumption is presented as a challenge to the research community.
ContributorsAmpofo, Prince Kwame (Author) / Tian, Xiaojun (Thesis advisor) / Kiani, Samira (Committee member) / Kuang, Yang (Committee member) / Arizona State University (Publisher)
Created2019