SPEAKER LIST

Dr. Tom McMurrough

Specific Biologics Inc.

Development of a next-generation therapeutic platform for in vivo human gene-editing

The over-arching goal of our project is to develop a robust next-generation gene-editing platform to repair the deleterious mutations that are responsible for genetic diseases such as Cystic Fibrosis and cancer. First-generation precision endonuclease technologies have been tremendous for in vitro gene disruption studies and ex vivo treatments, but there has been limited success at developing safe and effective in vivo human gene-editing therapies. To address these issues, we propose to package a highly specific RNA-guided dual nuclease technology (TevCas9), into liposomal delivery vehicles developed by Specific Biologics Inc. (SBI). By combining these technologies, we will create a powerful therapeutic platform that fulfills the target product profile for an ideal in vivo gene-editing platform. As proof-of-principal, we propose to target and repair the CFTR delta F508 mutation, a monogenetic mutation that results in Cystic Fibrosis (CF) and in which >85% of CF patients carry at least one copy of the mutation. CF was chosen as our model due to the high unmet medical needs of these patients and the suitability of SBI’s proposed liposomal (lipid-based) delivery system to target cells of the respiratory mucosa through nebulization. However, following our initial studies TevCas9 will be retargeted to other clinically relevant targets. 

Zach Gordon

Designer Microbes Inc.

Strain Domestication

Dr. Pratish Gawand

CEO, Ardra Inc

Production of Natural Ingredients through Synthetic Biology

 

Ardra is a Synthetic Biology start-up based in Toronto. Ardra’s focus is on production of natural ingredients for cosmetics and flavour and fragrance markets. With growing consumer preference for natural products, the demand for natural ingredients is on a rise. However, supplying natural ingredients at a stable price and quality is a challenge due to variability of the agriculture raw material. Synthetic biology can be used to solve the problem by engineering microorganisms to produce natural ingredients from sugars. Ardra has developed processed for producing natural ingredients such as butylene glycol (used in cosmetics) and leaf-aldehyde (used in flavors and fragrances). This talk will provide an overview of the development of butylene glycol technology with focus on metabolic engineering, fermentation process development and downstream processing.

Dr. Ian Silber

SGI-DNA

SGI-DNA BioXP 3200: Faster Results through Faster library and synthetic DNA production

SGI-DNA continues to increase the speed in which researchers can achieve results. In this presentation, we will discuss the BioXp™ 3200 System and other innovative technologies that can help researchers streamline the process from design to protein. The BioXp™ System is an automated genomic work station that quickly produces high-quality DNA libraries and clones on your bench. This flexible automated platform allows several key applications be performed hands-free. Paired with novel protein expression and developing DNA expansion technologies a researcher can go from design to transformation/transfection in less than 10 days.

Dr. Jordan Thomson

Ontario Genomics

Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Rebecca Shapiro

University of Guelph

​Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Trevor Charles

University of Waterloo

​Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Brendan Hussey

University of Toronto

​Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Kathleen Hill

Western University

​Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Mark Daley

Western University

​Panel Discussion: ​The Present and Future State of Synthetic Biology in Canada

Dr. Rebecca Shapiro

University of Guelph

Keynote lecture:

 

CRISPR-based functional genomic tools for complex genetic interaction analysis in fungal pathogens

Opportunistic Candida pathogens are the leading cause of fungal infections worldwide. Yet, comprehensive functional genomic analysis in these pathogens remains cumbersome. Here, we have developed a CRISPR-based toolkit for functional genomics in Candida, using canonical CRISPR-based mutations to map genetic interactions, and new strategies focused on CRISPR-based regulation of gene expression.

Thomas Hamilton

PhD Candidate, Western University

conCRISPR: Developing a conjugative tool to modulate microbial populations

 

The ability for CRISPR to target nearly any sequence of DNA allows it to be utilized to specifically kill bacteria. To prove effective, however, an efficient molecular delivery system is required. Motivated by our development of a dual-endonuclease, TevCas9, we developed and optimized a conjugation-based approach to deliver the CRISPR machinery and kill a bacterium of interest.

Dr. Isabel Desgagné-Penix

Professor, Université du Québec à Trois-Rivières

Diatoms for the production of valuable plant products.

To date, many fuels, pharmaceuticals, flavors and fragrances products are extracted from plants. These valuable plant products (PPs) are often produced in low quantities in planta and seasonal dependent growth, biotic and abiotic stresses can cause supply depletion. Organic chemistry can be used to produce synthetic PPs but the reactions are not always known, cost-effective or environmentally friendly. Thus, there is much interest in synthetic biology for metabolic engineering for developing microbial platforms to produce specific PPs. Microalgae are well-known systems for genetic engineering and suitable hosts for the reconstitution of complex plant pathways. Marine diatoms have gained much attention as they are expected to be a promising resource for the sustainable production of biofuels and biocompounds such as PPs. Recently genetic tools were developed to enable the creation of diatom Phaeodactylum tricornutum photosynthetic cell factories for the reconstitution of heterologous metabolic pathway. We developed a vector containing the genes encoding biosynthetic enzymes involved in PPs biosynthesis including the vanillin pathway. The eight genes were assembled into pairs in constructions with self-cleaving 2A peptide linker to coordinate expression. Each construction involved different combinations of promoters and terminator to enhance expression of transgenes and the plasmid can be propagated in P. tricornutum for long-term culture without rearrangements. The expression of transgenes mRNA was measured using RT-PCR analysis and corresponding enzymes were assessed using SDS-PAGE gel electrophoresis and Western blot assay. The production of vanillin in engineered P. tricornutum was determined using HPLC and LC-MS analyses. Preliminary results indicate that P. tricornutum is a promising platform for synthetic biology to produce PPs such as vanillin. This project is not only motivated to provide a proof of concept for the introduction of entire plant pathway in diatoms but also by consumer demand for products that are environmentally friendly, less expensive, and possess properties similar or superior with those generated by PPs.

Dr. Cintia Coelho

Brazilian Agricultural Research Corporation - Embrapa, Brazil. University of Brasília – UnB, Brazil.

Use of integrases to target genetic parts resulting in gene regulation in eukaryotic cells

 

 

Mayna Gomide (1,2), Thais Torquato (1,2), Leila Barros (1), Luciana Carvalho (3), Mariana Almeida (1), Rayane Lima (1), Marco Oliveira (1), Lilian Florentino (1), Andre Murad (1), Martin Bonamino (3), Cintia Coelho (1,2) and Elibio Rech (1)

 

1- Brazilian Agricultural Research Corporation - Embrapa, Brazil; 2- University of Brasília – UnB, Brazil; 3- National Cancer Institute – INCA, Brazil, Correspondence: elibio.rech@embrapa,br

 

DNA recombinant technology was a landmark for the development of the synthetic biology, which constitutes an intersection area between biology and engineering and should contribute to designing biological systems. The development of effective tools, which allow us to increase precision on gene regulation are essential for proper genetic manipulation. Current information has demonstrated effective control of the RNA polymerase flux using different integrases, capable to catalyse unidirectional inversion of DNA to turn on/off regulatory genes in prokaryotic cells. However, knowledge about the functionality of integrases in eukaryotic cells is still limited. Here we show the remarkable functional capability of bacteriophages serine integrases in plant, animal and human cells. A co-transformation plasmid system was utilized to evaluate integrases in A. thaliana protoplasts, bovine fibroblast and HK293T cells. The first plasmid contained the codon optimized integrase gene sequences under constitutive promoters. The second plasmid was the reporter plasmid that contains the gfp coding sequence gene in reverse complement orientation and flanked by the attB and attP sites of each integrase under constitutive promoter. Once the integrases were expressed, it would flip the coding sequence to its correct orientation and promoting GFP expression. The results proved that the coding sequence was flipped leading to the RNA polymerase flux through the DNA molecule at the forward orientation and the GFP expression, which was detected by fluorescent microscopy and flow cytometry. The coding sequence inversion was detected by PCR and sequencing analyses. We anticipate our results to be an initial point for development of more complex models of gene regulation in eukaryotic cells using integrases. 

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 for some species. 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 to clone, maintain, manipulate and transfer large DNA fragments (with an emphasis on high G+C content). We developed and optimized transformation protocols including electroporation and PEG-mediated transformation. Multi-host shuttle (MHS) vectors (pAGE and pBGE) were constructed, utilizing the minimal genome strain of S. meliloti, and identified oriVs from pSymA (pAGE) or pSymB (pBGE). The vectors contain origins of replication and selectable markers for S. meliloti, Escherichia coli, Saccharomyces cerevisiae, and the diatom Phaeodactylum tricornutum, as well as an origin of transfer necessary for conjugation. Significantly, we developed protocols for and demonstrated successfully the conjugation of the MHS vectors from S. meliloti to the aforementioned bacterial and eukaryotic microorganisms. Additionally, we demonstrated the potential to automate these conjugation protocols by performing conjugation from S. meliloti to P. tricornutum in a 96-well plate. The tools and protocols described here will facilitate the transfer and manipulation of large DNA fragments or whole chromosomes in S. meliloti for biotechnology applications.

Gurjit Randhawa

PhD Candidate

Analysis of Genomic Signatures in Our Current Era of Synthetic Biology

In synthetic biology, it is of immense importance to know whether a sequence structure is stable and its characteristic signature is maintained. Fast and reliable sequence comparisons facilitate rapid detection of synthetic systems and organisms, and tracking of genomic instability and decay in genetic signature. Multiple sequence alignment, a standard for sequence comparison, is computationally demanding and assumes conservation of contiguity between homologous segments. In the real world, collinearity is very often violated e.g. viral genomes show great variation in the number and order of genetic elements. We propose a machine learning based alignment-free method for genomic classification. Genomic sequences are represented as discrete numerical sequences and are treated as discrete signals. Discrete Fourier Transform (DFT) is applied on these signals to compute corresponding magnitude spectra. A pairwise distance matrix is computed by applying Pearson Correlation Coefficient (PCC) on all pairs of magnitude spectra. We train supervised machine learning classifiers on pairwise distance matrix- based feature vectors; new sequences are then classified using these trained classifiers. 3D visualization representing estimated dissimilarities among all sequences are generated using multidimensional scaling. We tested our methodology from Kingdom to Genus on more than 7000 mitochondrial genomes with average classification accuracy higher than 97%. Our ultra- fast method can quantitatively show the dissimilarity among any two sequences. Distinguishing genomic signatures and understanding the mechanisms that shape genomic signatures is relevant to understanding genomic integrity and instability. Our method is applicable to monitoring stability of synthetized genomes.

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.

Matt Berg

PhD Candidate, Western University

Mapping serine tRNA identity elements using mistranslation:

tRNAs read the genetic code allowing nucleic acid sequence to be translated into protein. Accurate translation requires the ligation of a specific amino acid to the 3’ end of each tRNA. This process is directed by nucleotides or base-pairs within the tRNA, known as identity elements, that are recognized by one of the ~20 aminoacyl-tRNA synthetases. For many tRNAs, the anticodon is the main identity element. This is not the case for serine tRNAs. Previously, we identified a serine tRNA containing a proline anticodon that mistranslates serine into proteins at proline codons. Using this mistranslating tRNA, we suppressed a stress sensitive allele of a co-chaperone protein in yeast, tti2-L187P. In this work, we used this mistranslation and suppression system to investigate the functional significance of nucleotides and base-pairs in tRNA-Ser. Further assessment was obtained by measuring the heat shock response induced by each mistranslating tRNA variant. We find the first and third nucleotide pairs in the acceptor stem play a role in tRNA function and a single base-pair in the D-stem is required function. Serine tRNAs also contain a unique variable arm between the anticodon and T-arms. We find both the length and the sequence of the variable arm are essential for tRNA-Ser function. Interestingly, incorporating the major alanine identity element—a G3:U70 base pair—into the acceptor stem of the serine tRNA abolishes function, suggesting that it is an anti-determinant. In addition, by taking advantage of the anticodon not being recognized by the synthetase during charging of the tRNA with serine and modulating tRNA function with different secondary mutations, we have generated tRNAs that mistranslate serine at both phenylalanine and arginine codons. Using this information, the serine tRNA can be engineered to incorporate serine at any codon with various efficiencies.

Samir Hamadache

Community Officer, SynBio Canada

Connecting Canada's Synthetic Biology Community:

SynBio Canada was recently launched to connect and foster the synthetic biology community from coast to coast. Through our website, synbiocanada.org, we showcase Canadian synthetic biology research, and host public profiles for researchers, industry leaders, and policy stakeholders. Interest from researchers and students so far has been incredibly encouraging, and we are now launching our first strategic planning cycle. We're looking forward to input from the Western synbio community, as we set our goals for the future.

Clara Fikry & Leah Fulton

Waterloo iGEM

Mixed populations are difficult to maintain for a variety of reasons. For instance, a difference in growth rates can result in one population outcompeting the other. Our team hopes to use synthetic optogenetic systems to dynamically control and maintain the growth of two populations simultaneously. To control bacterial growth, our systems will target MetE, an enzyme needed for the synthesis of methionine. Since methionine is essential for protein synthesis, bacteria can only grow if they have the MetE gene or if their environment is supplemented with methionine. By placing MetE production under the control of an optogenetically-regulated promoter, we can tune the growth of our bacteria. In order to control two different bacterial populations independently, each promoter will be regulated by a distinct optogenetic system. Thus, we can control growth of our two population by switching MetE production on and off with different lights.

Our system enables the growth of different microorganisms in the same culture. Hence, it opens several doors in biotech and research. It could improve the metabolic engineering of microbial populations used in the production of pharmaceuticals, biofuels, and other important materials. It could also allow researchers to explore complex interactions between microbes and investigate questions that could not previously be answered due to co-culturing limitations.

Luana Langlois & Ryan Cochrane

Western University

The Western Synthetic Biology Research Program: a student-led research initiative

The Western Synthetic Biology Research Program (WSBR)  is a student-led research initiative that seeks to provide fellow undergraduates a one-of-a-kind learning experience to driven and enthusiastic students in a research laboratory setting. By fostering a collaborative and friendly environment, students are liberated from the fear of failure and are equipped to explore science creatively. Our aim is to have an open laboratory space where students are provided with the resources necessary to venture out and explore the wonderful world of synthetic biology and biotechnology. Our diverse areas of actuation range from peer-led workshops, to journal club discussions, mentorship of fellow junior scientists, community outreach initiatives, to wet lab experimentation, data analysis, and troubleshooting. Students are able to acquire a wide array of soft skills, which are not only an asset to aspiring academics, but also highly marketable characteristics to those seeking to secure a position in industry upon completion of their degree. This presentation will highlight some of the recent advances of the newly-established initiative and provide an overview of our future directions.

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