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Plants synthesize and store oil mostly as triacylglycerols (TAG) in seeds that is transcriptionally controlled by WRINKLED 1 (WRI1), an APETALA2 (AP2) transcription factor. In contrast, avocado (Persea americana) mesocarp, which accumulates ~70% lipids by dry weight showed high expression of WRI2, along with WRI1 and WRI3. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Through in silico analysis, we identified that PaWRI2 has a single intact AP2 DNA-binding domain and lacks a C-terminal intrinsically disordered region (IDR3) and PEST motif, which likely results in a relatively stable protein, compared to its Arabidopsis ortholog. We further demonstrated that avocado WRI2 is functional, unlike Arabidopsis WRI2, and accumulates TAG when transiently expressed in Nicotiana benthamiana leaves. Additionally, co-infiltration of PaWRI2 with PaWRI1 and genes encoding for terminal step in TAG assembly, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and/or phospholipid:diacylglycerol acyltransferase 1 (PDAT1) also led to further increase in the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of > 40 fatty acid biosynthetic pathway genes revealed that several were distinctly up or down regulated by the expression of PaWRI2 and PaWRI1. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in nonseed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and nonseed tissues.

Climate change and its effects on our society is a steadily growing problem. In 2010, the industry sector accounted for more than 30% of the global greenhouse gas emissions. The chemical industry is one of the industrial subsectors responsible for the highest emissions of greenhouse gas. To reach the climate goals it is therefore urgent to find more sustainable options for production of chemicals in general. Synthetic biology and microbial cell factories are growing fields that have received much attention for inferring promising sustainable alternative production routes for various compounds. When it comes to microbial cell factories, cyanobacteria infer many advantages over heterotrophs. Cyanobacteria can for instance convert atmospheric CO2 into valuable compounds through photosynthesis using the light reaction and the Calvin-Benson cycle. In the present work, the freshwater cyanobacterium Synechocystis sp. PCC 6803 is metabolically engineered to produce 1,2-propanediol; an important chemical feedstock for which there is a great interest in finding a sustainable production route as an alternative to the current petrochemical one. Seven different constructs are designed for introduction and expression of a three-step heterologous metabolic pathway for 1,2-propanediol production. Two strains of Synechocystis are successfully engineered, with the heterologous pathway chromosomally integrated at the Neutral Site I through homologous recombination with an integrative plasmid targeting this genomic site. One of the three heterologous genes (mgsA) of the pathway was successfully translated as shown in a Western immunoblot. In a SDS-PAGE a band of 40 kDa was detected, corresponding to the size of both the sADH and YqhD enzymes.

Chromatin is decorated by a large array of biochemical modifications made to DNA and histone proteins. These modifications—and the broader organizational structure of chromatin—provide an important additional layer of information that is superimposed upon genome sequence and thus are widely referred to as the epigenome. The epigenome helps control which genes are expressed in a given context to produce the gene expression patterns that underlie the many different cellular phenotypes that arise during an organism’s development, and determine how these gene expression patterns are subsequently maintained for the life of an organism. The epigenetically heritable states are maintained and transmitted by self-propagating epigenetic mechanisms that persist in the absence of an initial stimulus. These epigenetic programs are generally thought to be controlled by core regulatory networks involving molecular writers and readers of chromatin marks. Guided by these principles, in this dissertation, we establish an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system consists of synthetic factors that can write and read m6A, and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we use our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits are able to program epigenetic functions de novo, conceptually validating “read-write” architectures. This dissertation outlines a synthetic framework for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells. / 2021-08-30T00:00:00Z

Thesis advisor: Abhisheck Chatterjee / The genetic codon expansion (GCE) is a technique that uses an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair to incorporate noncanonical amino acids (ncAA) into proteins, to enable more protein-based chemistry. In the past two decades, more than 200 ncAAs have been site-specifically introduced into proteins in E. coli, and facilitated studies of protein structures, functions and interaction with other molecules. Although a large variety of ncAAs are available for incorporation in the bacterial systems, significantly fewer ncAAs are accessible for incorporation in eukaryotic cells. An expanded GCE toolbox will be beneficial for numerous applications in eukaryotic systems. Currently, introducing ncAAs in eukaryotes predominantly relies on the archaeal pyrrolysyl tRNA/aaRS pair. Such a strong dependence on a single platform has precluded genetic encoding of many desirable ncAAs, including structural mimics of many important post-translational modifications. The work presented in this thesis first developed an engineered E. coli leucyl tRNA/aaRS pair to enable site-specific incorporation of citrulline, an important PTM, into proteins expressed in mammalian cells. This technology was used to reveal the role of citrullination on site R372 and R374 of PAD4. Additionally, aiming at genetically encoding more diverse ncAAs, all 20 E. coli derived tRNA/aaRS pairs were screened for their ability to suppress TAG and TGA in mammalian cells. This study revealed several tRNA/aaRS pairs that are suitable for ncAA incorporation in mammalian cells, including those selective for phenylalanine, lysine, arginine, serine and glutamine. Efforts are currently under way to engineer these pairs to genetically encode new structural classes of ncAAs. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Droplet microfluidics is a high-throughput platform capable of accelerating the screening and synthesis of biological and chemical systems. However, significant challenges to microfluidic design and fabrication limit its broad use. In this dissertation, I overview and present potential solutions to challenges in droplet microfluidic fabrication and design. First, I present a method for low-cost rapid prototyping of complex droplet microfluidic devices. By combining desktop micromilling and electrode integration with conductive ink, thermoplastic microfluidic devices can be produced with features as small as 75 microns that can be connected to external sensors and actuators. Such devices can be designed, fabricated, and tested within a day and are shelf stable for months. Next, I developed a droplet microfluidic component library using micromilling and conductive ink electrodes. This library is high-throughput, biocompatible, and consists of components for droplet generation, anchoring, reinjection, coalescence, picoinjection, capacitance sensing, fluorescent sensing, and sorting. These components were combined in complex workflows, specifically, in the development of multi-color droplet pixel arrays. Finally, a series of machine learning based design automation tools for droplet microfluidics were created. These tools are capable of predicting the performance and automating the design of single emulsion and double emulsion droplet generators across any fluid combination. Furthermore, two quality metrics were developed, versatility and flow stability, that provide important context on the behaviors of the suggested designs. These tools are the first of their kind in microfluidics, and can play an important role in shifting droplet microfluidic design away from the manual and iterative process it is today. These advancements in droplet microfluidic design and fabrication can set the basis to rethink the microfluidic development cycle. Predictable and reproducible design and fabrication of sophisticated droplet microfluidic devices would provide a next-generation automation platform for the biological and chemical sciences, running experiments orders of magnitude faster and more sensitive than current methods. / 2025-01-16T00:00:00Z

This thesis is concerned with presenting analyses regarding key ethical issues regarding and arising from the development and potential use of gene drive modified mosquitoes for the purpose of malaria elimination. Each of the chapters constituting this thesis offers a rigorously researched analysis which attempts to answer questions thus far unanswered in the academic literature. Chapter one explores whether the development and use of this technology can be fairly considered unethical in principle; concluding it cannot be. Chapter two explores the appropriate relationship between this technology and the precautionary principle, a prominent regulatory and governance principle which has been invoked as ostensible support for an indefinite global moratorium on all gene drive technology. The chapter concludes that the precautionary principle, at least as articulated by UNESCO, does not provide justification for a global moratorium on gene drive technology. In fact, the precautionary principle is likely unfit as a regulatory norm for some kinds of gene drive products and purposes. Chapter three was co-authored with Delphine Thizy, Global Stakeholder Engagement Manager for Target Malaria, one of the leading consortiums working on research and development of gene drive biotechnology for malaria control. Together we articulate the ethical principles selected to guide Target Malaria’s stakeholder engagement, as well as provide the rationale for their selection and expound upon some early lessons from their implementation. Chapter four offers an analysis with the goal of locating the ethically appropriate locus of political organization from which to seek permission for a gene drive modified organism release into the shared environment. The chapter considers the appropriateness of each of the following levels of political organization: consent of individuals, local communities, nation states, and international governance institutions. The conclusion arrived at, with some caveats, is that such a decision is most appropriately issued by a nation state. / Thesis / Doctor of Philosophy (PhD) / This thesis is concerned with presenting analyses regarding key ethical issues regarding and arising from the development and potential use of gene drive modified mosquitoes for the purpose of malaria elimination. Chapter one explores whether the development and use of this technology can be fairly considered unethical in principle; concluding it cannot be. Chapter two explores the appropriate relationship between this technology and the precautionary principle, a prominent regulatory and governance principle which has been invoked as ostensible support for an indefinite global moratorium on all gene drive technology. Chapter three articulates, expounds, and provides rationale for the ethical principles selected to guide stakeholder engagement by Target Malaria, one of the leading consortiums working on research and development of gene drive biotechnology for malaria control. Chapter four attempts to locate the ethically appropriate locus of political organization from which to seek permission for a gene drive modified organism release.

As the name suggests, synthetic biology designs new biology using human power, knowledge, and creativity. Biology is vast, complicated, and all-inclusive, and so is synthetic biology. I believe synthetic biology is the Utopia of biologists, chemists, physicists, material scientists, engineers,and computer scientists. It is a newly emerged and vastly growing field that can impact and improve our lives in many aspects. I dare to say that anything you see that is done by biology can, in the future, be done better by synthetic biology since, on top of having biology as a teacher and as a template, synthetic biology has the benefit of creative and rational design provided by the human brain. In a way, it is the next step in evolution. In this thesis, we worked on some yeast synthetic biology applications. We used engineered yeasts to create bandages to enhance and accelerate the healing of diabetic wounds, make biosensors for pathogenic bacteria and a small molecule metabolite (glucose) important in diabetic patients, and design a community of cells that could contain artificial intelligence. Chapter 1 gives a short introduction and background information regarding diabetes, wound healing, and advanced healing therapies. Chapter 2 is focused on engineering yeasts to secrete wound-healing proteins and in vitro and cell-based studies on the engineered yeasts and secreted recombinant proteins. Chapter 3 presents two wound dressings that contain engineered live yeasts as active ingredients. This chapter includes further in vitro and cell-based studies to assess the functionality of the designed dressings. Chapter 4 focuses on in vivo experiments to study the wound-healing properties of the designed live yeast dressings. Finally, Chapter 5 presents two other projects: one on live yeast biosensors and one on designing modular smart yeast communities that can do computation based on neural network algorithms.

Probiotics are live microorganisms that can confer health benefits to the host. They have long been consumed through fermented foods. While the specific mechanisms of probiotics are largely unclear, there is evidence that their beneficial effects may be attributed to the microbes’ ability to modify the gastrointestinal (GI) environment, to modulate host immune response, or to produce natural products that directly inhibit pathogens in the gut. With the increasing awareness of the important functions that the gut microbiota plays in affecting host heath, probiotics may no longer just stay as simple dietary supplements, but become a promising approach to disease management. With recent advances in synthetic biology, novel functions can be introduced into these “good” microbes to provide additional benefits. Genetically engineered bacteria have been developed to specifically target pathogens or effectively deliver therapeutics to the GI tract. However, there are significant limitations to the existing systems developed. For example, the engineered pathogen sensors largely rely on the similarity between the host and the pathogens, the therapeutics delivery systems are usually constrained by the molecular structures, and the majority of the works have been limited to laboratory settings. In this dissertation, I present a system we have developed based on a food-grade probiotic, Lactococcus lactis, and demonstrate a synthetic biology methodology that could be applied to build biosensors of other pathogens or environmental signals, as well as a generalizable peptide delivery vehicle to the GI tract. I will present my work in three parts. (1) The discovery of an effective antagonistic effect of L. lactis against the infectious diarrheal disease cholera, and elucidation of the mechanism with an infant mouse model. (2) The development of a diagnostic circuit in L. lacits that enables in situ detection of the pathogen and easy readout through fecal sample analysis. (3) The design of a generalizable therapeutic peptide delivery system utilizing the endogenous secretion pathway of L. lacits. Overall, my work exploits the natural and engineered benefits of the probiotic L. lactis and demonstrates its use in the intestinal disease diagnosis and therapy. / 2019-02-20T00:00:00Z