The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

Four experiments were conducted to investigate methods of producing transgenic bovine embryos entirely in vitro. Experiment 1 examined the effect of DNA microinjection at 11, 15 and 19 h after fertilization (haf) on survival rate and DNA detection frequency by polymerase chain reaction (PCR). There was no difference in transgene detection frequency between treatments (53% at 11; 50% at 15; 48% at 19 haf). Of all injected embryos developing to the morula or blastocyst stage after 7 d in culture, 89% tested positive for the presence of the transgene by PCR. Greater developmental efficiencies can be obtained when injection is performed early in pronuclear formation (7% (11/161) at 11; 4% (61159) at 15; 1 % (1/165) at 19 haf; p<0.05). Experiment 2 examined the effect of microinjection of DNA into the germinal vesicle (gv) of bovine oocytes on subsequent development and detection of the transgene. Injection of the transgene into the gv reduced developmental rates compared to controls (control=23% (89/384); non-injected=9% (23/250); GV injected=5% (12/259); p<O.05). Transgene detection frequency was 64% (37/58). Injection of bovine oocytes before fertilization results in viable embryos containing the transgene, although at low frequencies. Experiment 3 was designed to examine whether the frequency of microinjected DNA detection by peR In whole bovine embryos would decline over a 21 d culture period. At d 0, the transgene was detected in 100% (46/46) of embryos analyzed. At d 7, detection frequency was 84% (51/62) in viable embryos, at d 14 49% (18/37), and at d 21 38% (3/8). DNA detection frequency in microinjected bovine embryos by PCR analysis does not give a reliable indication of live transgenic birth rates until after 14 d in culture. Experiment 4 examined microinjected bovine embryos for their potential use as donor embryos in nuclear transfer, or cloning. There was no difference in development between embryos cloned from microinjected donor embryos and those from control donor embryos (injected=11 % (37/377); control=9% (7/81); p>0.05). Of the embryos developing from microinjected donors, 32% (12/37) were PCR positive. Microinjected embryos can be successfully used in a nuclear transfer program to produce more viable embryos, and the resulting embryos may be more reliably screened by PCR. The efficiency of producing viable bovine embryos positive for the injected gene may be increased by performing microinjection early in pronuclear formation, and entering the resulting embryos into a nuclear transfer program. / Ph. D.

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.

The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.

In my dissertation, I have worked on two distinct projects related to the immune system. The abstracts for the two projects that make up my dissertation work are below. In the first project (presented in Chapter 2), we study regulatory T (Treg) cells in chronic liver disease. Current dogma holds that Treg cells preserve tissue function in settings of inflammation and damage. Consistent with this, herein we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis, the key prognostic factor for patients – through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance has hitherto been unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner, also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, in the setting of NASH, we find that Areg from Treg cells promotes hepatocyte gluconeogenesis – through hepatocyte detection of fibrosis development and soluble mediators, including IL-6, produced by activated hepatic stellate cells – offering new insight into the cellular interplay of how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH. In the second project (presented in Chapter 3), we use engineered bacteria to produce chemokines in the tumor to promote anti-tumor immunity. Tumors employ multiple mechanisms to actively exclude immune cells involved in anti-tumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models – an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells – using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

No description available.

<p>Small molecule metabolites have important and diverse roles in every major cellular function. To study the activities of metabolites and the biological processes in which they are involved, it is important to be able to detect their levels within cells. Technologies that measure the concentrations of small molecules within the context of living, growing cells are highly advantageous but are challenging to produce. In this thesis, a novel class of intracellular small molecule sensors is produced, characterized and applied to address novel and relevant research questions. These sensors detect a specific target molecule within bacterial cells using RNA regulatory elements known as riboswitches and one of many possible reporter proteins. In addition to a project that yielded new methodology to create custom riboswitches, two projects that assess the capabilities of sensors that detect an active form of vitamin B12 are described. These projects present an abundance of data that provide novel insights into the transport and metabolism of vitamin B12 in <em>E. coli</em> cells. Overall, the results presented indicate that riboswitch-based sensors represent valuable and unique tools for the study of microbial biology. The thesis is concluded with a discussion that describes design strategies and several exciting potential applications for future riboswitch sensors.</p> / Doctor of Philosophy (PhD)

T lymphocytes engineered with chimeric antigen receptors (CARs) have shown remarkable success in the treatment of leukemias. Conventional CARs seek to recapitulate TCR and costimulatory signals through fusion of T cell signaling elements into a single receptor. The robust anti-tumor activity of CAR T cells is often accompanied by debilitating toxicities due to excessive T cell activation and cytokine production following infusion. Our lab has generated a novel chimeric receptor termed T cell antigen coupler (TAC), which is designed to engage native T cell signaling domains for cellular activation. In a murine xenograft model, we previously found that TAC T cells mediated rapid tumour regression in the absence of toxicities. Comparatively, CAR T cells elicited significant lethal toxicities to the mice due to reactivity against an unspecific antigen that resulted in excessive proliferation and cytokine production in vivo. Here, we report that TAC and CAR T cells have fundamentally different biology, both at rest, and during activation. TAC T cells were more sensitive to the context of stimulation compared to CAR T cells. Whereas TAC T cells can discriminate between antigen bound to a bead, or antigen present on a cell, CAR T cells do not make the same distinction and responds equally well to both. Compared to several different CAR constructs, TAC T cells are less prone to tonic signaling and T cell differentiation in the absence of antigen. These findings support that TAC T cells may pose a safety benefit as a cancer immunotherapy, due to its distinct biology from CAR T cells that enables them to require more stringent contexts for activation. / Thesis / Master of Science (MSc) / Cytotoxic T cells are also known as “resident killer” cells of the immune system, as they can seek and eliminate diseased or infected tissue, including cancer cells. However, cancer cells can evade elimination by T cells over time. Genetic engineering of T cells allows us to re-arm T cells against cancer cells. T cells isolated from a patient are genetically modified to recognize cancer cells specifically. So far, these modified T cells have been successful against several leukemias. However, the side effects of this treatment can be substantial and life-threatening, due to the massive reaction of the T cells against the cancer cells following infusion. We explore the biology of two different types of engineered T cells to better understand the interaction between T cell and tumour cell. Our results aim towards mitigating the side effects of T cell treatment, while investigating how we can improve its effectiveness for the future.

Genetic improvement of the potato through classical breeding has been limited by its tetraploid nature, the narrow genetic variability within cultivars, and interploidy barriers between tetraploid cultivars and diploid germplasm. Breeding at reduced ploidy levels has been proposed as a solution to these problems. Because of sterilities, somatic hybridization via protoplast fusion has been considered an alternative to sexual polyploidization for resynthesizing superior diploids from selected monoploids, and tetraploids from selected diploids and dihaploids. Successful application of somatic hybridization largely depends upon protoplast culturability and regenerability of a plant. The ability of callus formation and plant regeneration from protoplasts varies among plants. To understand the genetic basis for this variation, the mode of inheritance for protoplast culturability, defined as the ability to develop calli from cultured protoplasts, was studied in the diploid potato species, Solanum phureja. Based upon data from F₂ as well as from F₁ and backcross progenies, it was found that protoplast culturability in this potato species was controlled by two unlinked loci with dominant effect. In addition, there was quantitative variation for protoplast plating efficiency among culturable genotypes. Male sterility in cultivars of Solanum tuberosum ssp. tuberosum results from nuclear-cytoplasmic interactions. 'Donor-recipient' protoplast fusion and regeneration were conducted between a sterile S. tuberosum ssp. tuberosum cultivar, Russet Burbank, and fertile selections of S. tuberosum ssp. andigena which have a non-sensitive cytoplasm and were used as the cytoplasmic donor. Sixteen regenerated plants possessed nuclear background and chloroplast DNA of Russet Burbank. However, two of these regenerants had improved pollen stainability. The possible causes for the improvement of pollen stainability are discussed. In the last chapter, allelic polymorphism in a monoploid population derived from anther culture of a clone of S. phureja was assessed by isozyme electrophoresis. Fourteen monoploids and their anther donor were examined for six enzymes. No allozyme variation was detected in these plants. However, genetic variability among these monoploids was manifested by variations in some growth characters and general morphology. The limitation of enzymatic markers in detecting allelic polymorphism in these monoploids is discussed. / Ph. D.

The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events. / Master of Science