POSTERS

Jiarui Huang

McMaster University

The minimal pSymA symbiotic nitrogen fixation gene region of Sinorhizobium meliloti

Jiarui Huang, Zahed Muhammed, George C. diCenzo, Maryam Zamani, and Turlough M. Finan. Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada, L8S 4K1 

Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand the genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons essential for SNF: a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis identified that only four loci, accounting for <12% of the total sequences of pSymA and pSymB, were essential for SNF, and each replicon contained two SNF-essential regions. The two SNF-essential regions in pSymA are ~103 kb (402136 ~ 505335 nt) and ~52 kb (624863 ~ 677157 nt), respectively. Here we report progress towards the cloning of these two SNF-essential regions on a plasmid in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain. We are testing the SNF ability of this strain on alfalfa to determine if the SNF-essential regions from pSymA are sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with alfalfa.

Jason Kearsley

McMaster University

Isolation of a preliminary minimal N2-fixing symbiotic gene set of the pSymB megaplasmid in the host legume symbiont Sinorhizobium meliloti

 

Jason V.S. Kearsley and Turlough M. Finan

 

Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada L8S 4K1

 

Sinorhizobium meliloti is an important model organism for the understanding of the ecologically and agriculturally important N2-fixing symbiosis it forms with legumes. Its second megaplasmid, pSymB, contains several clusters of genes thought to necessary for symbiotic nitrogen fixation (SNF). The recent creation of an appropriate ΔpSymB background strain has enabled a gain-of-function approach to examine the sufficiency of several previously identified necessary regions on pSymB (representing 12% of the megaplasmid) for restoring SNF capacity. This work entailed the construction of plasmids that enabled Cre-loxP-mediated deletion of a large 325 kb region on pSymB that brought three critically identified SNF regions on pSymB adjacent to one another. Subsequent work utilized plasmids harbouring FRT sites that were employed in concert with Flp recombinase to excise the three SNF regions as a large 205 kb plasmid that was capable of capture by Escherichia coli. The ΦC31 integrase attP/attB site specific recombination system was then harnessed to transfer the captured SNF regions from pSymB back into the ΔpSymB background strain. This strain represents a preliminary minimal SNF gene set for pSymB in S. meliloti. Initial calcofluor-white fluorescence assays identified a substantial defect and delay in the production of the symbiotically relevant exopolysaccharide succinoglycan in said strain. It is suggested that genes outside of the main exo/exs cluster on pSymB may be more critical for succinoglycan biosynthesis than once thought. The symbiotic phenotype of the strain with Medicago sativa (alfalfa) is also explored.

Qin Dong

Western University

Designing DNA probes for whole genome tiling

Designing good DNA probes for whole genome tiling is a very challenging problem. The most difficult issue is ensuring high specificity of probes, which available programs do poorly. We introduce BestProbe, which computes better probes than the top programs, while running up to two orders of magnitude faster. BestProbe produces significantly better probes than those of Agilent for the human genome.

McShane McKenna

Western University

Phosphorylation-dependent inhibition of Akt1

Akt1 is a proto-oncogene that is over active in most cancers. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the roles of specific phosphorylation events in terms of Akt1 inhibition.

Yanrui Zhu

MSc Candidate, Western University

Cellular effects of mistranslating serine and proline tRNAs

Mistranslation can occur due to incorrect charging of tRNAs by aminoacyl-tRNA synthetases. Here, we use mutant proline and serine tRNAs to induce mistranslation in yeast strains containing gene deletions or mutations. We show that the genetic interactions of the mistranslating tRNAs are not identical but depend on both the amino acid being substituted and the amino acid being replaced.

Navaneeth Mohan

Western University

Detecting chaotic signatures in blood pressure fluctuations

Time series analysis of blood pressure recordings reveals the presence of low-dimensional, aperiodic, non-linear dynamics governing the blood pressure fluctuations. Further, the dynamics has a positive Lyapunov exponent. We are currently investigating the potential for such a non-linear dynamical analysis to improve current cardiac diagnostic tools.

David Wright

Western University

Genetic Code Expansion: Regulation of TrxR1 by acetylation

Thioredoxin Reductase 1 (TrxR1) defends human cells against oxidative stress and maintains the cellular redox balance. Using genetic code expansion to site-specifically insert both acetyl-lysine (acK) and selenocysteine (Sec), I was able to produce and purify human, AcK and Sec containing TrxR1 from E. coli. Acetylation increased redox activity and controlled TrxR1 oligomerization state.

iGEM Guelph

University of Guelph

The Heterologous Expression and Enzymatic Breakdown of Calcium Oxalate (Beerstone) by the Metabolic Enzymes FRC and OXC

Beerstone is the build up of calcium oxalate that precipitates on the inside of beer brewing machinery and storage equipment. The enzymes Formyl Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC) are used in the microbe Oxalobacter formigenes to naturally metabolize oxalate. Our project aims to characterize the activity and feasibility of these enzymes in the brewing industry.

Brendan Hussey

University of Toronto

Programmable T7-based synthetic transcription factors

We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein-protein interactions, which can be chemically or light induced, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli.

Maximilian Reed, Cullen Tielemans and Calvin Wong

University of Waterloo

Functional Amyloid as an Enzyme Scaffold

Though amyloid is often associated with non-functional states of proteins, the fusion of amyloid-forming amino acid sequences to enzymes presents the possibility of "anchoring" functional enzymes to the surface of amyloid particles. This facilitates the co-immobilizing of different proteins of an enzymatic pathway onto amyloid, decreasing diffusional limitations of the pathway in vitro.

Stephanie Brumwell

MSc Candidate, Western University

Development of Sinorhizobium meliloti as a host for cloning, storage and delivery of DNA to bacterial and eukaryotic microorganisms

The genetic manipulation of many organisms is hindered by a lack of available genetic tools. To circumvent this problem whole genomes or large DNA fragments of a donor organism can be transferred to a host organism for manipulation. Here, we report the development of protocols and genetic tools to establish
Sinorhizobium meliloti as a bacterial surrogate-host.

Dalton Ham

Western University

Development of a conjugative TevSaCas9 dual endonuclease system for use as a specific antibiotic

Traditional antibiotics target and kill bacteria non-specifically, resulting in the death of a spectrum of bacteria, including normal flora. To circumvent this, an antibiotic which is more specific in its mechanism is needed. By targeting bacterial genomes with programmable endonucleases, we can kill specific strains while leaving beneficial bacteria unscathed. Here we use TevCas9, a dual endonuclease, as a strain specific antibiotic. The purpose of this project is to replace the commonly used Streptococcus pyogenes Cas9 with a smaller Cas9 from Staphylococcus aureus. Because S. aureus Cas9 recognizes a longer PAM sequence and uses a longer guide RNA, we hope that this replacement will increase the killing efficiency of the system while simultaneously reducing the size of the protein.

Ali Heidari

Synthesis of 2-thiouracil and 2-thiothymine PNA for Fmoc-based synthesis of pseudo-complementary oligomers

Pseudo-complementary nucleobases are modified nucleobases which exhibit normal base pairing with unmodified complementary nucleobases; however, will not undergo base pairing with other pseudo-complementary nucleobases. 2,6-Diaminopurine, an adenine analogue, and 2-thiouracil (or 2-thiothymine), a thymine analogue, are two pseudo-complementary nucleobases which undergo normal Watson-Crick base pair

Maximillian Soltysiak

Western University

Development of a solid layer trans-kingdom conjugation protocol between Escherichia coli and Saccharomyces cerevisiae

A novel method was developed for trans-kingdom conjugation between donor Escherichia coli and recipient Saccharomyces cerevisiae that involves the coincubation of the two species while immobilized within an agar-selection media plate. Using the protocol, the effects on trans-kingdom conjugation efficiency was explored with different yeast replication origins and destination plasmid length.

Iqra Razzaq and Viola Halder

University of Guelph

The Study of Genetic Interactions in Candida albicans through the use of CRISPR-Cas9 and Golden Gate Cloning

Genetic interaction analysis is a powerful tool to assess if genes operate in singular, parallel, or independent pathways, and identify synthetic lethal interactions. We developed a CRISPR-based strategy to inactivate stress response genes in the fungal pathogen Candida albicans, and will use these mutants to assess genetic interactions and identify gene combinations involved in pathogen survival.

Rima Menassa

Agriculture and Agri-Food Canada

Plant-Made Virus-Like Particles for Protection of Piglets against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDv) is a coronavirus that causes disease and mortality to piglets particularly new-born piglets worldwide. Most vaccines used to combat the disease have been ineffective live attenuated virus vaccines. We have produced virus-like particles (VLP) displaying antigenic epitopes of the PEDv membrane protein (M protein) and envelope protein (E protein) in plants.

Adam Chin-Fatt

Developing an IgA biobetter antibody against E. coli O157:H7 with enhanced stability by rational design of a bovine Fc chain

The goal of this project is to demonstrate as a proof of concept that an immunoglobulin A(IgA)-nanobody fusion can be rationally designed to improve stability by engineering its Fc chain via supercharging and novel disulfide bond introduction. The project is notable because it presents a viable strategy for improving accumulation and value of the therapeutic without sacrificing efficacy.

Nathan Braniff

University of Waterloo

Optimal Experimental Design of Dynamic Perturbations and Sampling Schedules for Calibrating Models of Gene Expression

In this work we present methods for optimal experimental design that can reduce parameter uncertainty in models of gene expression. Numerical techniques from control theory and optimization are used to select optimal time-varying optogenetic induction patterns and sampling schedules. This results in increased informativeness of experiments and overall model accuracy.

Ondre Harper

MSc Candidate, Western University

Engineering self-assembling proteins to produce a safe and effective vaccine for Porcine Reproductive and Respiratory Syndrome Virus

Commercially available vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) provide some control over the virus but none are completely safe and effective. My goal is to use self-assembling protein nanocages as stable, non-replicating platforms to display antigens from PRRSV to the immune system in a multimeric, virus-like fashion.

Emily Clayton

Western University

Designing a novel assay to test for arogenate dehydratase activity in vivo

Phenylalanine (Phe) is an essential aromatic amino acid that is synthesized via two pathways: the arogenate or the prephenate pathway. The arogenate pathway requires the transamination of prephenate to arogenate by a prephenate aminotransferase (PAT), followed by a decarboxylation/dehydration of arogenate to Phe by an arogenate dehydratase (ADT). The prephenate pathway consists of the same two steps, but in reverse: a decarboxylation/dehydration of prephenate to phenylpyruvate by a prephenate dehydratase (PDT), followed by a transamination of phenylpyruvate to phenylalanine by a phenylpyruvate aminotransferase (PPAT). Arabidopsis thaliana has a family of six ADTs, all of which can convert arogenate to Phe. Interestingly two members, AtADT1 and AtADT2 can act as both ADTs and PDTs. PDT and ADT amino acid sequences are very conserved and currently cannot be used to distinguish between the two enzymatic functions. We can quickly determine PDT activity using an in vivo yeast complementation assay, however time consuming biochemical assays are required to detect ADT activity. It is therefore desirable to create a simple and cheap assay to test for ADT activity in vivo. We have developed a strategy based on a Saccharomyces cerevisiae PPAT knockout strain. Two PPATs were identified in S. cerevisiae: ARO8 and ARO9. As predicted, the ∆aro8aro9 homozygous strain is unable to synthesize Phe and will not grow without Phe supplementation. This ∆aro8aro9 strain was transformed with AtPAT to introduce the arogenate pathway. In the presence of a PAT, an ADT will complete the pathway to synthesize Phe in this yeast strain. This cheap and simple assay will be the only in vivo test of ADT activity that can distinguish between an enzyme with ADT/PDT activity, or only ADT activity.

Ryan Cochrane

MSc Candidate, Western University

The development of synthetic organelles for commercial and scientific use

Our goal is to create synthetic organelles as a way of transporting novel pathways, such as nitrogen fixation, back into organisms. Using two strains of algae, Phaeodactylum tricornutum and Thalassiosira pseudonana, different strategies were deployed to capture synthetic organelle genomes. In the future, various methods of delivering these organelle genomes into the cells will be tested.

Angelo Kaldis

Production of subunit vaccine candidates against Bovine Respiratory Disease pathogen Mannheimia haemolytica as an alternative to antimicrobials

The goal of this project is to produce an oral vaccine in plants which provides cattle with protection against infection with Mannheimia haemolytica, the predominant bacterial agent causing Bovine Respiratory Disease (BRD), or Shipping Fever, through mucosal immunity. Chimeric antigens were produced in Nicotiana benthamiana, Nicotiana tabacum and E. coli that will be tested in a mouse model of BRD.

Samuel Slattery & Helen Wang

PhD Candidate, Western University

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum

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 plasmid-based genetic tools described here are the next step towards improved manipulation of this species.

Benjamin Scott

PhD Candidate, University of Toronto

Coupling of Human Rhodopsin to Yeast Signaling Enables High-Throughput Characterization of Pathogenic Mutations

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with many GPCR mutations linked to human diseases. We have functionally linked human rhodopsin, a GPCR activated by light, to the yeast mating pathway. This system has allowed us to study patient derived mutations in high-throughput, to help understand the molecular basis for inherited retinal disease.

Jehoshua Sharma

University of Guelph

Using a CRISPR-Cas9 gene drive platform to characterize microbial persistence in the fungal pathogen Candida albicans

Our lab has designed a novel gene drive platform that utilizes the CRISPR-Cas9 complex in the fungal pathogen Candida albicans. The Cas9 gene drive creates an inheritance bias that was modified to cause large scale deletion libraries of genes with high efficiency. Here, we exploit this technology to characterize genetic interactions that regulate the survival of the pathogen Candida albicans.

Jeremy Lant

PhD Candidate, Western University

Understanding tRNA mutations and the 'hidden language' of protein synthesis.

Transfer RNAs ensure the faithful translation of RNA codons into protein. We have characterized mutations in tRNAs, from E. coli to humans, that break this relationship, causing mistranslation or overwriting of the genetic code, with surprisingly little impact on cellular viability. We discuss new perspectives on mistranslation, and possibilities for expanding the genetic code.

Chris Leichthammer

MSc Candidate, Western University

Development of Thermosensitive Endonucleases to act as Plasmid Kill Switches

Given recent developments in synthetic biology that enable engineering of large self-replicating synthetic elements for biological applications, an additional level of biocontainment is needed. Hence, we aim to develop and test genetic kill switches based on thermosensitive site-specific endonucleases that synthetic plasmids and chromosomes.

Coby Martin

Production of a Protein Subunit Vaccine for Mannheimia Haemolytica in Lettuce Chloroplasts

 

We designed a subunit vaccine against M. haemolytica, an infectious bacterium of cattle comprising a virulence factor and surface lipoprotein. Plant-based production of this protein will reduce cost by eliminating the need for purification. We transformed chloroplasts as a production platform for this subunit vaccine as they efficiently produce and accumulate bacterial proteins.

Alexander Roy

PhD Candidate, Western University

Make Cas9 Great (Again?): Development of TevCas9, for a more versatile and efficient genome editing tool

 

Cas9 has proven valuable for making targeted gene knock-outs. However, targeting is complicated by pseudogenes, which can lead to multiple disparate double-strand breaks and subsequent chromosomal translocations. TevCas9, by virtue of its dual nuclease activity, can be targeted to unique intron-exon junctions. We are producing TevCas9 variants with the ability to target any site in the genome.

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