Edinburgh_OG

Description: Are you using the right chassis? The continuously growing field of industrial biotechnology has incited the replacement of non-renewable processes with more energy-efficient ones. Central to this is the use of genetically modified organisms modified with recombinant DNA technology for a wide variety of applications, ranging from the production of fine chemicals and pharmaceuticals to fuels and bioremediation [1]. These technologies and applications are largely dependent on the use of well-characterised industrial host organisms including Escherichia coli and Sacharomyces cerevisiae. However, such strains are not always the optimal choice for particular processes due to inherent metabolic limitations or a lack of consensus between the engineered components introduced into the host from a heterologous organism for the particular bioprocess. Better Chassis for Better Application While the well-characterised E. coli and S. cerevisiae have a host of molecular biology tools available for engineering them since they have been domesticated (they have been vastly changed from their wild-type and adapted to laboratory conditions), they come with distinct limitations, such as the inability to make the post-translational modifications found in eukaryotic proteins, with a high potential for protein misfolding, aggregation and degradation, besides incomplete translation due to different patterns of codon usage to eukaryotes [2]. Using native producer organisms in biotechnology may lead to higher productivities, but these non-model organisms often lack well-characterised genetic engineering tools, which may present difficulties in optimising and controlling metabolic pathways. This presents a major barrier to the widespread use of these organisms as biofactories. Expand-Ed: Expanding Tools and Gene Editing for Non-Model Organism The Edinburgh Overgraduate iGEM team will address those challenges by domesticating the desired host strains, applying synthetic biology (synbio) genetic engineering standards to the design of protein expression and metabolic engineering systems in a number of non-model organisms and characterising the behaviour of these standardised systems in a number of host strains with high potential for industrial applications. Furthermore, although it has its limitations in bacteria, we will include the CRISPR-Cas9 platform to assess its utility in these organisms as it is a potentially useful tool to have a better control of genomic perturbations and maximise those optimal expression systems and metabolic pathways. Through our project, the path is being paved so that other participants in the synbio community will more easily access these organisms in the laboratory, accelerating the understanding of these organisms and increasing the list of strains that are suitable for the manufacture of relevant products, including those that address global conservation challenges. Our main aim is to open the opportunity of using a number of host chassis in synbio by characterising new DNA parts (e.g. promoters, reporter genes, terminators) that are necessary to produce proteins (through fluorescence observation) as well as the CRISPR-Cas9 technology, all through the modular cloning (MoClo) standard. Synechocystis sp. The first host organism we will be working on is the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria could be cell factories for biofuel production since they are able to fix carbon dioxide directly as their primary carbon source by using energy derived from sunlight and have higher growth rates [10]. This approach could help ease the greenhouse gases emissions since, currently, fossil fuels contribute to 98% of them [11] and, because they can be cultivated in non-arable land, they will not compete with food crops [12]. Rhodococcus jostii RHA1 Our second organism is the Gram positive bacterium Rhodococcus jostii. Such bacteria have applications in bioremediation as they are able to degrade polychlorinated biphenyls (PCBs), which are a variety of chlorinated compounds that, even though banned in the United States, they have been found in 500 of the 1,598 National Priorities List sites already identified by the Environmental Protection Agency (EPA) [13] and affect animal and human health by causing skin conditions, liver damage and even cancer [14]. Penicillium roqueforti Our third organism is the filamentous fungus Penicillium roqueforti that is used in the production of blue cheese. As previously mentioned, caution needs to be taken with this type of microorganism because of the production of mycotoxins. Unlocking new chassis: Risk and Safety Because these synbio developments are encouraged to be open-sourced [3], although biosafety/biosecurity measures have been successfully established for managing the model organisms and guidelines such as the Genetically Modified Organisms (Contained Use) Regulations 2014 exist [4], domesticating new strains comes with challenges that, if not addressed properly, they could be dangerous, risking both their function and their surroundings [5]. For instance, filamentous fungi are able to produce a wide array of important secondary metabolites including potential antibiotics and cancer drugs. Nevertheless, they also produce mycotoxins that can cause unwanted health and environmental problems, such as food and silage spoilage [6] and human disease (mycotoxicoses) [7]. Developing a risk assessment tool To enhance the biosafety and biosecurity by making researchers more aware of these potential problems, we are developing a risk assessment tool prototype that will allow researchers to mine the genomes of new species to identify toxic metabolites and genetic elements that might limit their immediate utility as industrial hosts. Based on the antiSMASH program [8], the prototype, having as an input the microorganism’s accession number, will display a list of the related secondary metabolites and their toxicity score/scale. This will enable the user to better assess the strains to be worked on in a more direct manner and will allow to have clearer targets for genome editing, either by knocking-down toxic secondary metabolites or enhancing beneficial ones. Following the previous example, by distinctly showing a filamentous fungus’ secondary metabolites and their toxicity levels, the practitioners would immediately know the potential targets for genome editing. By making this tool accessible – as a website, for example – as a community standard to those involved in the laboratory practice, the risks and benefits related to different strains could be communicated effectively, even across international borders and legal jurisdictions [9]. References [1] Nielsen, J., & Jewett, M. C. (2008). Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae. FEMS Yeast Research, 8(1), 122–131. http://doi.org/10.1111/j.1567-1364.2007.00302.x [2] Fakruddin, M., Mohammad Mazumdar, R., Bin Mannan, K. S., Chowdhury, A., & Hossain, M. N. (2013). Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli. ISRN Biotechnology, 2013(3), 1–7. http://doi.org/10.5402/2013/590587 [3] Evans, N. G., & Selgelid, M. J. (2014). Biosecurity and Open-Source Biology: The Promise and Peril of Distributed Synthetic Biological Technologies. Science and Engineering Ethics, 1065–1083. http://doi.org/10.1007/s11948-014-9591-3 [4] HSE. (2014). The Genetically Modified Organisms (Contained Use) Regulations 2014. Health and Safety Executive, 2002, 1–78. Retrieved from http://www.hse.gov.uk/pubns/books/l29.htm [5] Dana, G. V, Kuiken, T., Rejeski, D., & Snow, A. a. (2012). Four steps to avoid a Assess the ecological risks of synthetic microbes before they escape the lab ,. Nature, 483(1-March), 2012. [6] Filtenborg, O., Frisvad, J. C., & Thrane, U. (1996). Moulds in food spoilage. International Journal of Food Microbiology, 33(1), 85–102. http://doi.org/10.1016/0168-1605(96)01153-1 [7] Peraica, M., Radić, B., Lucić, A., & Pavlović, M. (1999). Toxic effects of mycotoxins in humans. Bulletin of the World Health Organization, 77(9), 754–766. [8] Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H. U., Bruccoleri, R., … Medema, M. H. (2015). antiSMASH 3.0--a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Research, 43(May), 1–7. http://doi.org/10.1093/nar/gkv437 [9] Marles-Wright, J. (2016). Better by Design, Safer through Practice. Synbio LEAP Strategic Action Plan, (January), 1–5. [10] Dismukes, G. C., Carrieri, D., Bennette, N., Ananyev, G. M., & Posewitz, M. C. (2008). Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Current Opinion in Biotechnology, 19(3), 235–240. http://doi.org/10.1016/j.copbio.2008.05.007 [11] Demirbas, A. (2009). Biofuels. Securing the Planet’s Future Energy Needs. [12] Dragone, G., Fernandes, B., Vicente, A. A., & Teixeira, J. A. (2010). Third generation biofuels from microalgae, 1355–1366. [13] Spaeth, K. (2011). Polychlorinated Biphenyls, 126(4), 217–218. http://doi.org/10.17226/19865 [14] Recio-Vega, R., Velazco-Rodriguez, V., Ocampo-G??mez, G., Hernandez-Gonzalez, S., Ruiz-Flores, P., & Lopez-Marquez, F. (2011). Serum levels of polychlorinated biphenyls in Mexican women and breast cancer risk. Journal of Applied Toxicology, 31(3), 270–278. http://doi.org/10.1002/jat.1672
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Updated at: 8/9/16