Bio-Rad qPCR Seminar- Training: Are you MIQE Compliant?
The MIQE guidelines have helped standardize the way research projects using qPCR are performed. qPCR is the gold standard technique for quantifying levels of RNA and DNA between samples. However, there remains a knowledge gap with respect to the application of rigorous methodology for each step of qPCR to assure reproducible results with accurate conclusions. Proper primer design and validation must be performed and the correct references genes must be selected. The final analysis of the resulting data is not trivial and there are wide variations between labs who typically use EXCEL spreadsheets created by individuals who may or may not understand how to convert the raw data to normalize results to statistical means, standard deviations and their significance with p-values.
Tuesday Sept. 12 11:30-12:30 BioSci Rm. 3110
Software training- 1:00-3:00 pm BioSci Rm. 3110
Characterization of pathways required for starvation longevity in Caenorhabditis elegans
Growth and development is controlled by specific sets of gene products (proteins and RNAs), that interact to form metabolic and developmental pathways. Mutations in these genes can result in abnormal growth of cells, leading to diseases such as cancer. "Phosphatase and tensin homolog" (PTEN) is one of the main proteins that control developmental and growth processes in humans. In the nematode Caenorhabditis elegans, DAF-18 is the homolog of PTEN. At the L1 larval stage, worms use an insulin-like signalling pathway to detect the availability of nutrients. DAF-18 is an antagonist of the insulin signalling pathway and is essential in maintaining quiescence during L1 arrest. Insulin-like peptides bind to the insulin receptor triggering a signal transduction pathway, which inhibits a transcription factor (DAF-16/FOXO) from entering the nucleus and activating the transcription of stress resistant genes. DAF-16 is located downstream of DAF-18. In L1 arrest, wild-type worms can survive up to 22 days without food. We and others have shown that daf-16 mutants show a decreased survival of 13 days in L1 arrest while daf-18 mutants can only survive up to 3-4 days. This discrepancy in time suggests that although DAF-18 is upstream of DAF-16, DAF-18 has additional functions essential in maintaining L1 arrest and L1 is, in part, independent of DAF-16 signalling. The aim of this study is to identify a potential alternate DAF-18 pathway, which works independently of DAF-16 and plays a crucial role in L1 survival. I predict that over- or under-expressing genes downstream of daf-18 in the alternate pathway will allow the daf-18 mutant worms to live longer than 4 days. I was able to isolate two suppressors through EMS screening, that can enhance the survival up to almost wild type even when DAF-18 is mutated. These mutants can help us to identify the proposed alternate pathway. I also studied the effect of DAF-2 on L1 arrest with somatic daf-18 rescue. The discovery of a new pathway will provide a better understanding of larval growth and development in C. elegans. Moreover, these findings could identify other genes that play major roles in control of cellular growth and the development of cancer.
Advances in genetic engineering empower the eukaryotic microalga Chlamydomonas reinhardtii for sustainable light-driven bio-production
Photosynthetic microalgae hold promise as green cell factories for sustainable light-driven bio-production processes. These organisms can be cultivated with freely available sunlight energy and CO2 as a sole carbon source, making them ideal chassis for sustainable production processes. Microalgae are already natural sources of many interesting bio-products including carotenoids, lipids, and polysaccharides. However, expanding the range as well as value of the compounds produced by microalgae through genetic engineering can increase the economic competitiveness of light-driven algal production platforms. In comparison to bacteria or yeasts, genetic engineering of eukaryotic microalgae has lagged significantly behind due to characteristically low transgene expression levels. Work in our research group has focused on engineering increased and reliable levels of nuclear transgene expression in the fast growing, Chlorophyceaen microalga Chlamydomonas reinhardtii, with the aim of generating heterologous bio-products from this photosynthetic host. We employ several levels of optimization in transgene design, followed by synthetic development of nuclear cassettes in order to ensure robust expression of target constructs. Recently in our group, the development of a fully synthetic and optimized nuclear gene expression vector (pOptimized) further increased the range of possibilities for nuclear encoded transgene expression in C. reinhardtii. We have applied this system for numerous tasks including: recombinant protein production via secretion into culture media, in vivo localization of fluorescent reporter-fusions for pathway elucidation, and metabolic engineering for novel isoprenoid generation. Our high-throughput workflow allows a level of reliable nuclear transgene engineering, which was previously unattainable in this host. This presentation will overview our recent successes in engineering nuclear transgene expression in C.reinhardtii for numerous applications, with a highlight on the production of non-native isoprenoids.
Acute starvation and its transgenerational toll: linking epigenetic modification to germline integrity
During the winter of 1944/45 occupying Nazi troops rationed a small population of Dutch inhabitants to approximately 1000 calories/day. Later in life, the children that were conceived during this period were found to have an abnormally high frequency of a number of disorders ranging from obesity to schizophrenia. Differences in the DNA methylation patterns between siblings suggest that the resulting disorders may be epigenetic in nature and that the experience of this acute starvation was written into the chromatin of these individuals, and presumably into their heritable genome within the germ cells. The factors that mediate this heritable defect in the germline remain unclear, so we use the germline stem cells of C. elegans as a model to understand how such environmental stresses impinge on the chromatin. We have shown that quiescence and germline stem cell integrity are dependent on AMP-activated protein kinase (AMPK) in specific starvation-dependent developmental contexts, and animals that lack this activity exhibit transgenerational reproductive defects. Our data suggest that the inappropriate regulation of chromatin modifiers and small RNA regulators are responsible for the reproductive defects that occur uniquely in AMPK mutants in response to acute energy stress/starvation. We are currently in the process of identifying the targets of AMPK that mediate these changes, while focusing on the regulation of small RNAs and their potential role in directing these epigenetic phenomena.
Improving freezing tolerance in temperate cereal crops: what can we learn from Brachypodium model species
The effects of low and freezing temperatures on plants have long been the subject of intense research, as they limit crop productivity worldwide. To date, many studies have described cold-acclimation in cereal crops, the process by which an exposure to low, non-freezing temperatures induces freezing tolerance. The accumulated data demonstrate that cold acclimation requires the expression of different but overlapping suites of genes (multigenic traits). In addition, the regulation of low temperature responses in plants has been shown to involve dynamic epigenetic changes at specific loci. My research aims at understanding the chromatin mechanisms that control agronomically important traits such as freezing tolerance in cereals. To study chromatin dynamics we are using monocot Brachypodium species as genetic model systems. These plants are close relatives of wheat and barley and are appealing for molecular studies due primarily to their small genomes and the relative ease in which they can be transformed. The latter is crucial to our work in the lab as transgenic plants allow us to isolate and analyze the function of specialized chromatin modifying genes involved behind this targeted response. In this talk I will discuss these mechanisms and highlight some projects currently underway in my lab at McGill that we hope will ultimately lead to strategies for the improvement of important cereal crops.
The Complex and Enigmatic World of Freshwater Algal Viruses
As well as being agents of infectious disease, it is now widely accepted that viruses are intimate partners in life that have shaped the evolution of cellular organisms and have played an ongoing role in Earth’s biogeochemistry. With respect to the latter, the discovery almost 30 years ago that viruses infected marine and freshwater primary producers, including species of eukaryotic algae, contributed to the emergence of virology as its own discipline within aquatic sciences. My research program is centered on freshwater algal viruses with the broad goal to understand how they influence aquatic primary production and food web processes. To that end, my students and I have explored freshwater algal virus ecology by studying their biodiversity and seasonal dynamics, persistence in the environment, and relative importance to phytoplankton mortality. We have learned that diverse algal viruses can be found in various freshwaters, but as in marine environments, freshwater algal virus communities are surprisingly dominated by a single group. We have also discovered that algal viruses can form seasonally persistent ‘seedbanks’, and some can overwinter in a frozen pond remaining infectious until their host abundances reach levels we presume are necessary for ongoing virus production. Additionally, the fact the we were able to detect diverse viruses in Lake Erie sediments suggests that sediments are an important refugium that could allow some phytoplankton virus to escape seasonal bottlenecks when they suffer high rates of destruction. With respect to our studies of virus-mediated mortality, we have developed experimental methods to examine phytoplankton mortality at a species, or taxon level. An unexpected finding from this work was the observation that in some cases, viruses appeared to counteract the effects of grazing mortality by stimulating an alga’s growth, perhaps through the liberation of resources from other lysed cells, or by reducing competition. Finally, an additional focal point of my research program is our isolation and ongoing characterization of a newly discovered algal virus. Molecular characterizations of this virus in the lab and field have revealed its complex evolutionary history as well as its puzzling ecological dynamics. Overall, my research has helped demonstrate that, despite their furtive nature and borderline status as ‘living’ microorganisms, freshwater algal viruses are critically important members of freshwater ecosystems.
11:30-12:30 BioSci Rm. 1102
*** And Free Pizza Meet & Greet 12:30-1:30 in BioSci 3rd floor lunch room (Rm 3406)
Characterization and knockdown of a family of ice-binding proteins from freeze-tolerant grasses
Plants are exposed to environmental stresses that threaten their growth, reproduction, and survival. At sub-zero temperatures, the recrystallization of ice in the apoplast results in cellular dehydration, disruption of the plasma membrane and cell death. Certain plants from temperate regions produce ice-binding proteins (IBPs), which protect plants by adsorbing to ice crystals and modifying their growth. Using the model crop, Brachypodium distachyon, we have identified seven novel IBPs and characterized their in vitro ice-binding activity, in planta induction, and post-translational modification. We have also shown the ability of these proteins to attenuate bacterial ice-nucleation activity. Additionally, we have generated the first IBP-knockdown in any organism, allowing us to finally confirm the importance of these proteins in membrane protection and freeze-survival. Expression of a number of IBPs from the perennial ryegrass, Lolium perenne, in Arabidopsis thaliana has localized IBPs to the apoplast, with the identification of one IBP isoform that is non-classically secreted. IBPs provided freeze protection to A. thaliana in a localization-dependent manner, with an enhanced phenotype associated with the expression of multiple isoforms. This research highlights the potential utility of IBPs for the generation of freeze-hardy crops, where previous transgenic studies have shown limited success.
Characterizing the roles of Arabidopsis calmodulin-like protein, CML39, in hormonal regulation of early seedling development and fruit formation.
Calcium (Ca2+) is considered among the most ubiquitous and versatile second messengers in eukaryotes. Cytosolic Ca2+-oscillations are evoked by environmental stimuli such as biotic or abiotic stresses and developmental cues. In turn, these Ca2+signals are detected by Ca2+-binding proteins, termed Ca2+ sensors, that help coordinate physiological responses by binding to and regulating the activities of various proteins. Calmodulin (CaM) is an evolutionarily-conserved eukaryotic Ca2+ sensor involved in many signal transduction pathways. Interestingly, plants possess large families of unique Ca2+sensors related to CaM and known as calmodulin-like (CML) proteins. Several CMLs have been implicated in developmental and stress-response signalling but the roles of most CMLs remain unknown. We recently reported the importance of Arabidopsis CML39 in early seedling establishment (Bender et al 2013, Plant J: 76:634). CML39 knock-out (KO) mutants display developmental arrest in the absence of exogenous sucrose. Our ongoing phenotypic analysis of cml39 knock-out plants has identified several additional developmental abnormalities in these mutants. In comparison to wild-type plants, cml39 mutants display perturbations in response to exogenous hormones, altered fruit morphologies, and unusual germination properties. Qualitative and quantitative studies describing the phenotypic characteristics of cml39vs wild-type plants are presented. In addition, a putative interacting partner (CML39IP) of CML39 was identified through yeast-two-hybrid screens. Here, we present preliminary data for the delineation of the interaction domain along with the phenotypic analysis of KO mutants of CML39IP.
Follow the chemistry: Making and breaking odd bonds with enzymes.
Microbes are an incredible source of diverse enzyme chemistries. This reflects an unrelenting evolutionary pressure to develop catabolic and biosynthetic pathways that allow microbes to survive, and even thrive, in diverse environments. Such pathways allow microbes to extract nutrients from a wide array of substrates, as well as defend themselves from other organisms through 'chemical warfare'. With recent advances in whole genome sequencing and bioinformatics, the microbial world has become a happy hunting ground for the enzymologist who is interested in finding unusual enzymes. This seminar will focus on two programs that are driven by genomics driven discovery of new enzyme reactions. The first involves the enzymatic cleavage of the C-P bonds found in phosphonates, while the second involves the enzymatic synthesis of C-F bonds. Recent advances in these two programs will be presented.
11:30-12:30 BioSci Rm. 3110