Juan Luis Suárez
Title: Synthetic Biology: A New Dynamic to Modern Human Identity
Bio: Juan Luis Suárez brings an interesting research perspective and expertise to Synthetic Biology through investigation and reflection on impacts to human identity and human evolution. In addition to being a professor in both Digital Humanities and Computer Science, Juan Luis Suárez is also the director of the CulturePlex lab at Western University. He is a well-established multidisciplinary collaborator investigating 16th– and 17th– century globalization, current sectors affected by digital innovation, cultural analytics, and digital humanities. He has also developed software tools for creative industries and was the co-founder of Milao Language, a language learning start-up. Further, Suarez was recently named Associate Vice-President (Research), adding imperative representation from social sciences, arts, and humanities to the Research Western leadership team.
Title: You Want an Honours Biotechnology Grad from Fanshawe College in Your Lab.
Bio: Cheryl Ketola completed her Ph.D. at Western University in Gene Expression (Zoology) and a post-doctoral fellowship in Insect Cell Biology (Zoology). She joined Fanshawe College in 2004 and was instrumental in the design and launch of the Honours Bachelor of Applied Technology (Biotechnology) program. In addition to her position as professor, she has coordinated the program since its inception.
Cheryl and her colleague Dr. Michael Jennings are actively engaged in applied research into the biological and chemical properties of healing honey. Their collaborations are diverse. They maintain an active and interactive research focus with Dr. James Brennan and his group at IT Sligo. The marketing side of healing honey is also investigated through a relationship with Mr. Conor Daly of H!VE, Active Irish Honey in Donegal, Ireland. Here in Canada, in partnership with iB4e technologies Inc., Cheryl and Michael are creating a database to house the national and international honey data. Currently they hold an NSERC grant which employs 2 Biotechnology students to assist in the collection and analysis of honey. Components of the honey research are incorporated into the upper level courses in the Biotechnology program.
Title: Mutagenesis in New DNA Sequence Contexts
Bio: Kathleen brings expertise in the analysis of mutagenesis to the burgeoning field of Synthetic Biology. Kathleen is an Associate Professor in the Department of Biology, Adjunct Professor in the Departments of Computer Science and Ophthalmology at Western, and Associate Member at the Lawson Health Research Institute. Her research team discovers mutation signatures and identifies mutagens and mutational mechanisms relevant to development and aging, phenotypic diversity, vision loss, cancer and the evolution of genes and genomes. The team’s discoveries are significant in understanding how mutations arise and appreciating the dynamic complexity that mutations contribute to fitness. The team’s results provide important baselines of spontaneous mutations used to assess environmental exposures and compromises to genome integrity. Current collaborative research identified signatures of genome organization that constrain mutational diversity. With the rise of synthetic DNA sequences that may knit together different genomic signatures in sequence organization, Hill’s team seeks to model, predict and observed the dynamics of mutagenesis in these new sequence contexts.
Title: Methane to Bioproducts
Bio: Trevor Charles is a bacterial geneticist with a research program in plant-microbe interactions, functional metagenomics, and bacterial genome engineering for bioproducts. Following B.Sc. Microbiology at University of British Columbia, he obtained his Ph.D. in Turlough Finan’s lab at McMaster University (symbiotic nitrogen fixation) and did postdoctoral work in Gene Nester’s lab at University of Washington (Agrobacterium). He held a faculty position at McGill University before moving to his current position at University of Waterloo in 1998. He is co-founder and CSO of the company Metagenom Bio Inc., which applies metagenomic and microbial community analysis to challenges in the agriculture and mining sectors.
Title: Mobilizing Ontario’s SynBio Community
Bio: Ihor Boszko joined Ontario Genomics as Vice President, Business Development in October 2016. In this role, Ihor leads the activities of the business development team, focussed on supporting early stage companies in a variety of industry sectors to solve business challenges by leveraging commercially-relevant genomic technologies and innovations.
Prior to joining Ontario Genomics, Ihor spent nearly 15 years in the in vitro diagnostics (IVD) industry developing, commercializing and partnering innovative molecular technologies. Most recently, he worked at Xagenic Inc, joining at seed stage in February 2011 to help build this 10 person start-up into a commercial success. Under the guise of a “business development” role, Ihor led marketing and corporate development, helped the company raise >$50M in Series A & B venture capital financing, built out a facility and wore many other hats.
Previously, Ihor was Manager of Business Development at Luminex, where his work included licensing, technology assessment, strategic planning, and competitive intelligence. Prior to that, Ihor was with Tm Bioscience for 5 years, where he was initially part of the R&D team that developed the core technology, and then helped to commercialize that same technology in a Business Development role.
Ihor studied undergraduate genetics at Western, holds a MSc in molecular biology from McMaster University and an MBA from Queen’s University.
Title: Novel MRI Reporter Genes for In Vivo Molecular Imaging
Bio: Hello! My name is Nivin Nyström and I received my Bachelor of Science (Honours) in Genetics. I then joined the Department of Medical Biophysics at the University of Western Ontario as a graduate student, co-supervised by Dr. Timothy Scholl and Dr. John Ronald at the Robarts Research Institute. I am the recipient of a 2017/18 NSERC CGS-M Scholarship and of the 1st Place Oral Presentation Award at London Imaging Discovery Day 2017. My current research involves equal parts magnetic resonance imaging (MRI) and molecular biology, toward the development of novel reporter genes that would allow for detection of subcellular events in living animal models with high spatial anatomic information. My broader interests lie in the development of novel biotechnologies that involve creative, unique approaches to complex problems. Accordingly, I am an avid follower of research coming out of the J. Craig Venter Institute in Maryland, the Zhang Lab at MIT, as well as the Gambhir Lab at Stanford University. In my free time, I enjoy refining my jiu jitsu at open mat challenges, and more generally, exercising to keep healthy.
Title: Mathematical Modelling of the CcaS/CcaR Optogenetic System
Bio: Nathan is a PhD student in the Ingalls Lab in the Department of Applied Mathematics at the University of Waterloo. His research focuses on mathematical modeling in systems and synthetic biology. His current interests are in the use of optogenetic systems for the dynamic control of cellular processes. He is working on optimal experimental design to support the development of kinetic models of these systems as well as for optimal dynamic control of enzymatic activity, with applications in bioprocesses.
Nathan received his BSc in computer science from Queen’s University in 2013. He earned an MSc in computer science from Queen’s University where he worked on bioinformatic methods for identifying genes linked to prostate cancer metastasis, and an MMath in computational mathematics from the University of Waterloo where he worked on numerical methods for simulating fluid flow in gravity currents.
Brendan J. Hussey and David R. McMillen
Title: A programmable T7-based transcriptional activation system in bacteria
Bio: David McMillen is an Associate Professor in the Department of Chemical and Physical Sciences at the University of Toronto Mississauga. He did his PhD in the University of Toronto’s Faculty of Engineering, and postdoctoral work at the Center for BioDynamics at Boston University, with James Collins and Nancy Kopell. His research combines experimental and theoretical techniques to study systematic methods of controlling cellular behaviour, and ways of putting cells to work to create low-cost solutions to real-world problems. Examples of applied projects include efforts at developing systems to: detect disease microorganisms and respond to counter them; create microorganism-based biosensors for disease diagnosis; and designing a probiotic to help those suffering from inflammatory bowel diseases.
Title: Evolving mistranslating tRNAs through a phenotypically ambivalent intermediate
Bio: Matt Berg is currently a PhD candidate in Biochemistry at Western University having completed his Honors BSc at Western in Biochemistry and Cell Biology. His projects in the Brandl lab are centered on examining how cells misinterpret the genetic code, the consequences of mistranslation and its applications. Specifically, his work has focused on mutations in tRNAs that result in altered decoding. These mutations can be to the anticodon or to key nucleotides which direct charging of an amino acid to its cognate tRNA. Either can result in ambiguous decoding during translation, where one codon is decoded as two different amino acids across the proteome. Mistranslating tRNAs have a number of potential uses in synthetic biology. Furthermore, by studying these tRNAs, Matt will be in a position to test models for how the genetic code evolved and examine the relationship between mistranslation and disease.
Title: The Western Synthetic Biology Research Program: passion and innovation, a student-led initiative towards creating opportunities in Synthetic Biology
Bio: Undergraduate education in North America is second to none. The sheer number of possibilities, state-of-the-art facilities, and exceptional faculty creates a formidable environment for conducting cutting-edge research, a worldwide standard. Students often overlook the potential for taking part in research and distinguished academic involvement, an unfortunate result of a herd mentality that dictates the norm and hampers innovative ideas from being developed. Fortunately, with just enough passion and intellectual curiosity, along with freedom for creativity – and to some extent ingenuity – it is possible to grow beyond the confines of the trivial into the extraordinary. The Western Synthetic Biology Research Program (WSBR) was founded out of a desire to create opportunities for such keen students to be involved with research, providing a creative outlet for scientific innovation. Formerly known as the UWO iGEM team, Synthetic Biology at Western has grown to encompass not only bench work and practical research, but also an outreach program to high school students, scientific article scrutiny during journal clubs, gaining attention of both faculty and fellow students to the growing field of Synthetic Biology. WSBR escapes the ordinary by harbouring the possibility of self-driven learning, allowing the pursuit of knowledge out of personal interest rather than imposed restraints, thus making the students the protagonists and ultimate masters of their own education.
Reza Saberianfar and Rima Manassa
Title: Production of a bovine/camelid antibody against Shiga Toxin producing Escherichia coli in plants
Bio: Reza Saberianfar completed his B.Sc. in Biology at TMU of Tehran where he received a 20/20 for his thesis. He came to Canada to do his M.Sc. at Dr. Karagiannis’s lab at Western. His research on characterization of a gene involved in cell cycle regulation resulted in two publications. He joined Dr. Menassa’s lab for his Ph.D. to study the mechanism of protein body formation and their use as a tool in biotechnology. He published five first author papers including research articles, a book chapter, and an opinion article, and co-authored three other research articles during this time. One of his research articles were featured on the cover of the Plant Biotechnology Journal. Reza received several awards and scholarships during his studies including Ontario Graduate Scholarship, Dr. Uchida fellowship in life sciences, Dr. Lumsden graduate fellowship, Dr. Arnold fellowship, and Dr. Roth memorial award. His PhD thesis received the Best PhD Thesis award (Dr. Detwiler), and was nominated for the Governor General of Canada Gold medal by the faculty of science, also nominated for Canada’s Distinguished Dissertation Award by the Biology department. Reza has received several best oral presentation awards in international, national and regional meetings. Currently, he holds an NSERC visiting fellowship at Agriculture and Agri-Food Canada.
Turlough Finan KEYNOTE SPEAKER
Title: Genomic manipulations towards a minimal set of symbiotic genes for the bacterium Sinorhizobium meliloti
Bio: Turlough Finan is a professor of Biology at McMaster University, Hamilton, Ontario. His interest in secondary replicons originated during his B.Sc and M.Sc in Microbiology under the supervision of Kieran Dunican, National University – Galway, Ireland. He obtained his PhD in 1981 under the supervision of Carl Jordan, Microbiology Department, University of Guelph, Canada. Following a year at the Connaught Research Institute studying the neutralizing antigens of Polio virus he then performed postdoctoral studies on Sinorhizobium under the supervision of Ethan Signer in the Department of Biology, Massachusetts Institute of Technology. He teaches undergraduate and graduate courses on Microbiology, Molecular Genetics and Environmental Microbiology. For over 30 years he has examined genomic and metabolic aspects of the interaction between Sinorhizobium meliloti and alfalfa. Initial studies focussed on the detection and analysis of symbiotic loci and expanded to the biology of the 1.7 Mb pSymB replicon, including carbon and phosphate metabolism.
Title: Creating Symbiotic Relationships Between Academia and Industry to Advance Synthetic Biology Research
Bio: Preetam Janakirama is currently a Staff Scientist at Designer Microbes Inc (DMI). DMI is a Synthetic Biology company that offers novel innovative solutions to enable the engineering of microbes to produce food, DNA storage technologies, next-gen fuels and medicine. He has a Bachelor of Science degree in Life Sciences and a Master’s degree in Microbiology from Bangalore University. He later received a PhD in Plant Molecular Biology from Western University. After completing his PhD, he was an NSERC Visiting Fellow at the London Research and Development Centre of Agriculture and Agri-Food Canada in London, Ontario with Dr. Krzysztof Szczyglowski. During his tenure there, he investigated the role of a symbiotic gene in nodule development in the model legume plant Lotus japonicus. He joined DMI in March 2017 after finishing his fellowship.
Title: Ardra – A biotech start-up story.
Bio: Jonas studied biochemistry at the university of Tuebingen, Germany before he moved to Zuerich, Switzerland for his PhD. During his PhD work at the ETH Zurich, he focused on E. coli’s central metabolism and explored the possibilities of using C-1 carbon sources as a feedstock. After his PhD, he moved to Canada and joined the laboratory of Prof. R. Mahadevan at the University of Toronto and investigated the production of industrially relevant chemicals using genetically modified microorganisms. When Ardra, which was formed during his time as a postdoctoral fellow in Prof. Mahadevans laboratory, applied for the biotech accelerator program Indiebio in San Francisco, USA he joined the company as the technology lead. During the time in San Francisco his focus was on advancing the existing technology to produce 1,3- butanediol and expanding the product and patent portfolio. In late 2016 the decision was made to move Ardra back to Toronto and continue development from there. Currently, the company operates out of Jlabs Toronto and after some period of business development and fundraising, Jonas started to work in the laboratory again.
Title: Mathematical Modelling of the CcaS/CcaR Optogenetic System
Abstract: A consistent theme in both synthetic and systems biology is the accurate characterization of dynamic behaviour of cellular processes. Using previously published time series flow cytometry data from the Tabor lab, we parameterized a novel mechanistic model of the CcaS/CcaR system. Our modelling work suggests that an essential dimerization reaction may be involved in signal transduction, which has not previously been reported. We also performed a model comparison between the previous phenomenological model and our more mechanistic descriptions of the system.
Title: Predictive modelling of a batch filter mating process
Abstract: Quantitative characterizations of horizontal gene transfer are needed to accurately describe gene transfer processes in natural and engineered systems. A number of approaches to the quantitative description of plasmid conjugation have appeared in the literature.
In this study, we seek to extend that work, motivated by the question of whether a mathematical model can accurately predict growth and conjugation dynamics in a batch process. We used flow cytometry to make time-point observations of a filter-associated mating between two E. coli strains, and fit ordinary differential equation models to the data. A model comparison analysis identified the model formulation that is best supported by the data. Identifiability analysis revealed that the parameters were estimated with acceptable accuracy. The predictive power of the model was assessed by comparison with test data that demanded extrapolation from the training experiments.
Matheus Sanita Lima
Title: Unraveling organelle genome transcription using publicly available RNA-sequencing data
Abstract: Organelles exhibit incommensurable diversity in genome content, structure and size together with unique gene expression mechanisms governed by organellar and nuclear apparatuses. Next generation sequencing (NGS) has been producing unprecedented amounts of genomic and transcriptomic data that could be better used to study organelle genome transcription. That is because most of the RNA-sequencing (RNA-seq) data are still generated to investigate only nuclear transcription, even though organelle reads could be mined from those experiments. Here, we investigated organelle genome transcription of 89 plastid-bearing protists using already publicly available RNA-seq data. Our RNA mapping analyses unravelled pervasive (near-full to full) transcription of mitochondrial, plastid (including cyanelle) and nucleomorph genomes. At least 85% of each of those genomes is being transcribed, including large portions of non-coding (intergenic and intronic) DNA. These results reiterate the idea that organelles transcribe all their genomic material first and then apply heavy post-transcriptional processing to their polycistronic transcripts. If the allegedly non-coding regions are producing functional non-coding RNAs, organelle genome size variation might provide raw material for new regulatory RNAs that could have shaped the evolution of plastid biogenesis and trophic mode determination
Title: Molecular and evolutionary origins of the unusual co-enzyme specificity of Quinone Reductase 2.
Abstract: Quinone Reductase 2 (NQO2) is a FAD-linked enzyme that catalyzes 2-electron reductions of quinones and other electrophiles. NQO2 is highly unusual in that it uses the conventional nicotinamide cofactors, NADH and NADPH, very inefficiently compared to small nicotinamide cofactors such as dihydronicotinamide riboside (NRH). This is in stark contrast to its “sister” enzyme, NQO1, which uses NAD(P)H efficiently, supporting its clear enzymatic function in the cellular response to oxidative and xenobiotic stress. The unusual coenzyme specificity of NQO2, combined with the apparently small or non-existent amounts of such coenzymes in cells, suggests that NQO2 may have non-enzymatic functions such as intracellular redox signalling.
We are interested in the molecular origins of the unusual cofactor specificity of NQO2. Comparison of active site residues in NQO2 with those in NQO1 was used to select several for mutation, which yielded a 70-fold increase in catalytic efficiency with NADH as coenzyme. This “local” approach to engineering the coenzyme preference of NQO2 will be supplemented with a more “global” approach by examining the evolution of the two enzymes. Whole genome sequences indicate that NQO1 is present in all vertebrates, while NQO2 is absent in fish and amphibians, but present in mammals, birds, and reptiles. Therefore, NQO2 is limited to the amniotes, which are vertebrates that diverged roughly 312 million years ago to lay their eggs on land or retain them within the mother. The goal of this analysis is to characterize particular enzyme sequences at key branch points in the evolutionary pathway in order to understand which residues throughout the protein contribute to the cofactor specificity.
Title: Harnessing Asaia sp., a commensal bacterium of vector mosquitoes, for in vivo production and delivery of dsRNA in mosquito control
Abstract: Mosquitoes are considered among the deadliest animals on Earth due to the diseases they carry, such as malaria, yellow fever, dengue fever, and Zika. The Sterile Insect Technique (SIT) is a pesticide-free method of controling pest insects through the production and release of sterile males. For SIT to be used in mosquito control, some difficulties associated with this technology, such as lack of efficient sex-sorting and reduced mating competitiveness of the radiated male insects must be overcome. RNA interference (RNAi), a double stranded RNA- (dsRNA) mediated gene silencing technique, could provide an alternative method to producing sterile male mosquitoes, by targeting both female viability and male fertility transcripts simultaneously in developing mosquito larvae. Due to the high production costs and the time required for large-scale dsRNA synthesis and purification, bacterial symbiont-mediated RNAi is being considered as a means of providing mosquito larvae with the necessary dsRNAs to produce sterile males in insect production facilities. Asaia bacteria densely colonize the gut of major vector mosquitoes, and in this study, we have isolated a strain of Asaia with the aim to engineer it to produce and deliver dsRNA to the host mosquito larvae. Lambda-red mediated homologous recombination was first used to create an RNaseIII-deficient strain of Asaia. A genetic construct with two opposing constitutive strong promoters capable of transcribing dsRNA to any targeted gene was designed, synthesized and ligated into a broad host range plasmid backbone for in vivo/in vitro expression of the dsRNA within the bacteria. This Asaia strain along with its dsRNA expression vector could potentially be used for mosquito control purposes as well as reverse genetic studies.
Title: Producing a virus-like particle for protection of piglets against Porcine Epidemic Diarrhea Virus
Abstract: Porcine epidemic diarrhea virus (PEDv) causes disease and mortality to piglets worldwide. Most vaccines used to combat the disease have been ineffective live attenuated virus vaccines. The goal of this project was to produce a plant-made virus-like particle (VLP) displaying antigenic epitopes of the PEDv membrane protein. To obtain sufficient protein and subsequent VLP assembly, we used an elastin-like polypeptide fusion with the membrane protein. The resulting protein accumulated up to 0.8 mg/g of fresh leaf weight when transiently expressed in Nicotiana benthamiana. Virus-like particles were observed when the membrane protein was overexpressed, but also when the membrane protein was co-expressed with the envelope protein. In the latter case, the VLPs were slightly larger in size. This represents the first time coronavirus-like particles have been made in plants, and is a first step towards a plant-made oral vaccine for PEDv.
Title: Production of subunit vaccine candidates against Bovine Respiratory Disease pathogen Mannheimia haemolytica as an alternative to antimicrobials
Abstract: Bovine Respiratory Disease (BRD), also known as Shipping Fever, results in high economic loss of feedlot cattle in North America. It is a multifactorial disease which develops as the result of transportation stress, mixing of cattle, and bacterial agents – predominantly Mannheimia haemolytica. Current controls against BRD are aimed at bacterial pathogens. Antimicrobials are employed for therapeutic and non-therapeutic uses. There is also an injectable vaccine available but it only provides systemic immunization. The goal of this project is to produce an oral vaccine in plants that provides cattle with protection against infection of M. haemolytica-induced BRD. Constructs containing chimeric M. haemolytica antigens for both nuclear (Nicotiana benthamiana) and chloroplast expression (Nicotiana tabacum) have been produced and expressed. Each chimeric construct contains an N-terminal Cholera Toxin B (CTB) element for mucosal immunogenicity fused to a modified virulence factor by a short, flexible linker. An antigen of interest (AOI) from M. haemolytica is fused to the C-terminus with a second, larger flexible linker. AOI’s were chosen and engineered to consist of important epitopes and immunogenic sites of proteins from M. haemolytica. Constructs were transiently expressed in N. benthamiana and targeted to 5 different subcellular compartments. Recombinant protein accumulation levels of chimeric AOI-1 peaked in the E.R. at 2.9% of total soluble protein or 0.4 g/kg of fresh weight. AOI-2 protein failed to accumulate to detectable levels in any of the compartments tested in this transient expression system. Both AOI’s are currently in the early stages of tobacco chloroplast transformation.
Title: The role of PprA in extreme genome reassembly of Deinococcus radiodurans
Abstract: The bacterium Deinococcus radiodurans is distinguished for its ability to survive atypical exposures to DNA-damaging agents, particularily exposure to ionizing radiation. D.radiodurans can tolerate hundreds of DNA double-strand breaks (DSB) throughout its genome without any loss of viability due to very efficient DNA repair pathways. The mechanism of error-free genome reassembly is not well understood but several Deinococcus-unique repair factors have been identified. The protein PprA seems to be particularily important for DNA repair as it is upregulated in response to various DSB-inducing agents, is essential for recovery from DSBs, and has no evidence of homology beyond the Deinococcus genus. Here we show that PprA functions by oligomerizing into large protein filaments and by interacting directly with DNA. Filament formation and DNA binding are completely independent functions but are both essential for accurate genome reassembly. In order to facilitate understanding of the molecular mechanism on an atomic scale, the X-ray crystal structure of PprA fromD.radiodurans was solved to a resolution of 2.1 A. Subsequent structural determination of PprA fromD.peraridilitoris and D.deserti revealed an identical fold to that of D.radiodurans as well as protein-protein interactions that may be relevant in the context of higher-order filament formation.
Title: Production of a recombinant protein vaccine for Mannheimia haemolytica in lettuce chloroplasts
Abstract: The cattle industry worldwide is ravaged by bovine respiratory disease (BRD), a bacterial disease caused by Mannheimia haemolytica. Recent efforts to design vaccines against M. haemolytica focus on a virulence factor, leukotoxin, in addition to surface lipoproteins. Plant-based protein production is a safe and inexpensive alternative to traditional methods. Edible vaccines deliver antigens to pharyngeal tissues, which can provide local immunization against M. haemolytica to prior to its progression into the lungs. In this project, M. haemolytica antigens will be produced in lettuce chloroplasts as a candidate edible vaccine for BRD. This endeavor necessitates the adoption and optimization of lettuce growth and transformation and will thereby support future work with transplastomic lettuce.
Title: Characterizing the function of terminal uridyl transferase 1
Abstract: Terminal uridylyltransferase 1 (TUT1) is a human poly(A) and poly(U) polymerase which adds adenine or uridine tails to the 3’ ends of RNA substrates causing strand stabilization. TUT1 is activated via phosphorylation of serine and threonine residues by upstream kinases involved in cellular stress responses. It polyadenylates pre-mRNAs in a novel splicing and tailing mechanism that stabilizes the strands for translation, specifically for genes involved in the oxidative stress response and apoptotic initiation. TUT1 polyuridylates U6-snRNA, the central RNA strand involved in intron splicing, by restoring a four uridine-long tail on its 3’ end in order to stabilize it, thus preserving the integrity of its splicing functions. TUT1’s adenylation and uridylation activity thus make it a key player in protecting the cell from DNA damage (and consequent cancerous mutations) and ensuring proper intron splicing. TUT1’s nucleotide preference between ATP and UTP is unknown, as is the extent of the role played by residue phosphorylation on its activity. We aim to assess TUT1’s nucleotide preference for these nucleotides via radioactive kinetics assays using non-phosphorylated (WT) TUT1, as well as phosphoserine and phosphomimetic glutamic acid mutants, incubated with [α-P32] UTP and ATP and substrate RNAs. This will elucidate both the nucleotide preference of the polymerase and the influence played on its activity by post-translational phosphorylation. Doing so will provide further insight towards the activation of TUT1 in response to cellular stresses and the consequences of its activity on cell survival.
Title: RNA specificity of Gld2 is defined by site specific phosphorylation
Abstract: The cellular levels of active microRNAs are crucial to maintaining cellular homeostasis and are influenced by 3’-end adenylation or uridylation. However, these two catalytically similar reactions have different outcomes: adenylation stabilizes miRNAs while uridylation silences or destabilizes miRNAs. Improper nucleotide additions have been shown to lead to diseases such as breast cancer and hepatocellular carcinoma. The enzymes responsible for these nucleotide additions are known as terminal nucleotidyltransferases (TNTases). Gld2 (germline development 2) is a minimal TNTase involved in modifying mRNAs and miRNAs in the cytoplasm and has been implicated in the stabilization of the miRNA miR-122, an essential component for the propagation of the Hepatitis C virus. Previous reports presented conflicting data of Gld2 as an adenylyltransferase or uridylyltransferase, suggesting two opposing roles. Our data has shown that Gld2 is, in fact, a bona fide adenylyltransferase involved in stabilizing RNAs and that absence of a crucial histidine residue in the active site imparted a preference for ATP over UTP. We have also shown that Gld2 activity and RNA specificity is regulated by serine phosphorylation in the disordered N-terminal domain. This is similar to the regulation of RNA polymerases through the C-terminal domain and this form of regulation may explain the lack of RNA binding domains in Gld2. Interestingly, fluorescence anisotropy assays only show slight differences in miR-122 binding by phosphomimetics. It is also interesting to note that while alanine substitutions at the studied serine sites greatly reduced catalytic activity, RNA binding was not as greatly impacted. These results provide a glimpse into the regulation of Gld2 activity and RNA specificity and distinguishes the cellular role of Gld2 from other TNTases that act as uridylyltransferases.
Title: Differential Expression of Two RNA Editing TUT Isoforms May Play a Role in Breast Cancer Development
Abstract: Terminal uridylyltransferases (TUTs) play a role in RNA regulation by adding untemplated nucleotides to the RNA 3’end, marking RNAs for degradation. Human terminal uridylyltransferase 4 (TUT4) plays a role in the uridylation of the miRNA let-7, known to regulate oncogenes. TUT4 mediated let-7 monouridylation increases let-7 levels, whereas polyuridylation decreases let-7 levels. More specifically, TUT4 protein expression has been shown in vivo to be upregulated in breast cancer cells, reducing levels of let-7 and allowing for cell proliferation. TUT4 exists as two splicing variants, the full length TUT4 (1644 aa) and a second, shorter isoform containing only the first 719 amino acids, termed sTUT4. Very little data is available for sTUT4, as all current studies focus on the characterization of full length TUT4. We here describe the initial characterization of sTUT4. Our data suggests that TUT4 and sTUT4 expression is altered in more aggressive cancer cell lines and this pattern may play a role in the deregulation of miRNA levels in cancer cells. Recently, we have shown through in vivo studies a decrease in sTUT4 in late stage breast cancer cell line MDA-MB-231. sTUT4 was previously believed to be inactive due to mutations in its catalytic domain, but in vitro radioactive studies have shown sTUT4 to be active, with increased polyuridylation over monouridylation activity. This leads us to believe that the shorter isoform plays a novel role in breast cancer tumorigenesis pathways and is a potential therapeutic target in treating late stage cancer.
Title: Transcriptional regulation of cell-type specific expression in the Arabidopsis root
Abstract: Characterizing transcription factor interactions with their corresponding binding sites is crucial for understanding how gene expression is regulated by DNA sequence. A more comprehensive understanding of this process could have benefits in synthetic promoter design and creation of genetically modified organisms. Herein, the promoters of genes exhibiting cell-type specific expression within a single layer of the Arabidopsis root are analyzed to identify cis-regulatory motifs implicated in cell-type specific expression. De novo motif prediction identifies multiple motif candidates overly represented in the promoter sequences of co-expressed genes specific for epidermal, cortex, and endodermal expression. Several endodermal specific putative motifs are further analyzed for positional biases and tested in planta. A priori mapping of known cis-regulatory motifs catalogued in publicly available databases is also performed. Results show that cell-types contain different statistically significant enrichment patterns of both predicted and known cis-regulatory motifs. These results will help future research in designing cell-type specific synthetic promoters.
Stephanie Brumwell and Tony Huang
Title: Development of Sinorhizobium meliloti as a surrogate host for genome-scale engineering
Abstract: Cloning and engineering DNA is essential in molecular biology. Recently, there is growing interest in the cloning and manipulation of whole chromosomes or large DNA fragments as it has widespread applications in the study of organisms that are difficult to culture, the development of synthetic pathways or genomes, as well as the capability to introduce a modified genome back into an original (recipient) organism. Currently, Saccharomyces cerevisiae can be used as a surrogate-host system to clone and manipulate whole chromosomes or large DNA fragments of a donor genome. However, S. cerevisiae cannot maintain large DNA fragments with G+C content >40% without additional engineering, and direct transfer (via conjugation) of these fragments to a target organism is not possible. Therefore, we aim to develop Sinorhizobium meliloti as a bacterial surrogate host to clone, maintain, manipulate and transfer large, high G+C content DNA fragments. Recently, a strain of S. meliloti was constructed lacking the pSymA and pSymB replicons, resulting in a 45% reduction of its genome. Additionally, the replication origins of pSymA and pSymB were identified and can be used to support the replication of very large plasmids. We will report on the development of multi-host shuttle (MHS) vectors, utilizing the minimal genome strain of S. meliloti and identified oriVs, to facilitate the cloning, maintenance and manipulation of large DNA fragments. The MHS vectors contain origins of replication and selectable markers for S. meliloti, Escherichia coli, S. cerevisiae, and the diatom Phaeodactylum tricornutum, as well as an origin of transfer necessary for conjugation. Conjugation protocols were developed and we demonstrate the successful transfer of these vectors from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum, and report the conjugation efficiency.
Title: The development of synthetic organelles for commercial and scientific use.
Abstract: The goal of this project is to create synthetic organelles to eventually introduce novel pathways, such as nitrogen fixation, in organisms. Using two strains of algae, Phaeodactylum tricornutum and Thalassiosira pseudonana, four different strategies will be deployed to capture a synthetic organelle genome. T. pseudonana’s relatively small mitochondrial genome, ~48kb, was the first genome to be captured and transformed into E. coli using a PCR approach. Next steps will require this approach and others, such as transformation-associated recombination cloning, to be used for the remaining organelle genomes. In the future, various methods of delivering these organelle genomes into the cells will be tested prior to introducing novel pathways.
Title: Spatial statistical tools for mutation cluster detection tested using the case of F1 heterozygosity and a cancer model.
Abstract: Genome sequences can be synthesized with different susceptibilities to mutation and thus different evolutionary potentialities. Increased mutation rates have been observed in association with high heterozygosity in Arabidopsis. Here, we investigate this phenomenon in Mus musculus by analyzing the spatial distribution between heterozygous single nucleotide polymorphic (SNP) genotypes and de novo copy number variants (CNVs) using the Mouse Diversity Genotyping Array. First we studied in inbred parental mice (0 to 5% heterozygosity) and first filial (F1) hybrid mice (19 to 53% heterozygosity). Higher heterozygosity in the F1 mice does not appear associated with more CNVs or proximal CNVs. Heterozygosity is not consistent with proximal CNVs during mitosis. Parental mice with higher heterozygosity had more CNVs, consistent with elevated mutagenesis in meiosis. Second, we studied heterozygosity levels and de novo CNVs in a cancer model with elevated mitosis, elevated heterozygosity and aggressive metastasis (MMTV-PyMT: Rhamm-/-). We observed a 2-fold increase in the number of chromosomes that show evidence of heterozygosity associated with more and proximal CNVs in Rhamm-/- mice compared to wild-type mice. However, the majority of chromosomes did not show clustering between heterozygous genotypes and CNVs. Our observations prompt investigation into elevated mutagenesis associated with heterozygosity and sequence context in meiosis. Mutagenesis in the context of meiosis and the production of germ cells requires intensive study to predict integrity of synthetic DNA contexts.
Title: Isolated DNA Vs. Synthetic DNA and Patent-eligibility.
Abstract: The biotechnological invention was not a patentable subject matter anywhere in the world before 1980. For the first time in 1980, the US Supreme Court in Chakrabarty case (1980) decided that the biotechnological innovation can be patented if it meets the requirements of the patent. Then the European Union enacted the Directive on the Legal Protection of Biotechnological Inventions (1998). It was the first written law which made DNA patentable. The Canadian Supreme Court also in Harvard College case (2002) decided that the lower life form (DNA) is a patentable subject matter. However, everybody was not convinced with the argument that DNA is a patentable subject matter. There were objection and criticism against the idea of DNA patentability. Again in 2013, the US Supreme Court in Myriad case (2013) considered this issue and decided that isolated DNA is just a discovery and not a patentable subject matter. The US Supreme Court in this case further stated that synthetic DNA is patentable. The issue of discovery with respect to isolated DNA has been settled, but the issue of ‘description’ has not been settled in Myriad case. The issue of ‘description’ equally applies to synthetic DNA as well. Abstract, claim and description are the basic and fundamental requirements of a patent application. The poster discusses the issue: is patent the right type of intellectual property protection to synthetic DNA?
Title: Identifying non-histone lysine methylated proteins via synthetic peptides and AP-MS/MS
Abstract: Post-translational modifications (PTM) are vital regulators of protein function and homeostasis. The role of dynamic regulations of non-histone lysine methylated proteins (NHKMP) recently began to be recognized in DNA damage repair, apoptosis, and transcriptional pathways. A perturbed NHKMP network can lead to diseases such as cancer. Our goal is to identify components of the NHKMP network to fully understand its importance in healthy versus diseased states. Currently, the best method of enriching NHKMP can only identify 72 NHKMPs. The main impediment to identifying more NHKMPs is the lack of a high-coverage method: we devised one to capture NHKMPs in vitro via affinity purification coupled with tandem mass spectrometry (AP-MS/MS). A mixture of five naturally occurring lysine methyl-binding domains (KMBD) was used. A biotinlyation site was incorporated into each KMBD: they were chosen based on their overlapping specificities to achieve maximum coverage of lysine methylated peptides. Each individual KMBD specificity was mapped out using substrate and permutated peptide arrays. A total of nine different KMBDs were tested: the results from the permutated array demonstrated that five of them have distinct specificities. We performed affinity pulldown using those five KMBDs on standard peptides and trypsinized HEK293 cell lysates; the peptides were identified via MS. We identified over 200 distinct non-histone lysine methylated peptides with various methylation states corresponding to 148 NHKMPs from HEK293. The amount of NHKMPs that we have identified in a single run is unprecedented: this allows us to gain valuable insight into components of the lysine methylome network.
Samuel Slattery and Helen Wang
Title: Plasmid-based tools for generating designer Phaeodactylum tricornutum strains
Abstract: With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. Finally, we constructed a plasmid encoding a putative eight-gene vanillin biosynthetic pathway that was propagated in P. tricornutum over four months with no evidence of rearrangements. Whole-plasmid sequencing was used to track mutation accumulation over time, with the majority of mutations occurring after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.
Thomas Hamilton and Chelsey Ryan
Title: A CRISPR-based system to modulate microbial populations
Abstract: Without a doubt, the impact that pathogenic bacteria can have on human health is unquestionable. Recently, a great deal of research has considered the effects that variations in human microbiomes can elicit on our health. These results suggest that a system with the ability to specifically modulate these microbiomes is required. To do this we aim to develop a plasmid-based system to manipulate microbial populations using CRISPR, and our engineered dual-endonuclease, TevCas9. We have shown that our engineered plasmid can be conjugated from Escherichia coli to Salmonella typhimurium using the mobilization plasmid pTA-mob. Additionally, we have shown that the creation of a self-conjugating version of this plasmid can conjugate with up to 3000-fold higher efficiency. We were then able to show that our system is effective at killing S. typhimurium conjugates with up to 99.5% efficiency. As we seek to further develop the system, we intend to show its function in an environment more like that of a true microbiome. We also wish to explore the versatility of the system as it applies to different microbes, and to improve the conjugation efficiency of the plasmid.
Title: The awesome power of tRNA engineering: Encoding phosphothreonine in E. coli and tRNA-dependent mistranslation in human cell culture.
Abstract: The last decade has seen remarkable advancements in the field of genome editing, giving us unprecedented control over the fate of living cells, and empowering a new tier of precision medicine unlike anything we could have achieved with small molecule inhibitors. While the ability to easily cut-and-paste DNA into genomes is a remarkable step forward, it is still limited to the confines of our genetic code. Meanwhile, protein translation scientists have been making great progress toward obtaining such control over the translated proteome. Translation-level editing provides several advantages. Firstly, it enables direct encoding of protein modifications including phosphorylation, acetylation, and more. Translation-level editing can also be employed in a less obtrusive manner than typical genome editing, wherein mutations can be suppressed or overwritten without permanently altering codons in open reading frames. Lastly, translation-level editing has the potential to encode engineered amino acids which do not exist in nature, such as fluorescent groups, ‘clickable’ groups, purification tags, and many more. My research focuses on engineering tRNAs and translation systems to encode phosphorylated amino acids (pSer, pThr), as well as investigating tRNA-driven mistranslation in human cells, with the dual purpose of investigating how tRNAs may behave as disease modifiers, and whether mistranslation could be deployed to suppress deleterious mutations. Here the first ever RNA-guided, sense codon recoding system for phosphorylated amino acids is reported in E. coli, as well as the first ever evidence of tRNA-driven mistranslation in human cells.
Title: Production of chimeric secretory IgAs against Enterohemorrhagic Escherichia coli in plants
Abstract: E. coli O157:H7 is the most prevalent pathogenic Entero-Hemorrhagic E. coli (EHEC). Cows are the main reservoir of this strain. The pathogenicity of E. coli O157:H7 is due to the presence of an attachment and effacement system as well as their ability to produce Shiga toxin. The main contributor to E. coli O157:H7 virulence is their ability to colonize the gastrointestinal tract. The proteins Intimin and Tir are the main factors allowing colonization by binding to each other and keeping the cell attached to the epithelial cell. Our solution is the passive immunization of E. coli O157:H7 by using secretory IgAs to target Intimin within the gastrointestinal tract of cows, and to prevent colonization. We used camelid antibodies (VHHs) because of their higher binding capacity and stability compared to bovine IgAs, and fused them to bovine IgA-Fc. Secretory component (SC) and joining chain (JC) were also co-expressed with IgA-Fc-VHHs to make the fully-assembled sIgA in Nicotiana benthamiana. We also generated transplastomic Nicotiana tabacum plants expressing camelid VHH(s). In each case we use two VHH candidates, “Naïve” from a non-immunized llama, and “High Affinity” another from an immunized llama with Intimin.