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− | <h2 class="section-heading"> | + | <h2 class="section-heading">Description Summary</h2> |
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+ | <br><br><br> | ||
<img src="https://static.igem.org/mediawiki/2016/0/05/T--Edinburgh_OG--Matin_Back_Interlab.jpg" width= "95%"> | <img src="https://static.igem.org/mediawiki/2016/0/05/T--Edinburgh_OG--Matin_Back_Interlab.jpg" width= "95%"> | ||
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<h3> Motivation<h3> | <h3> Motivation<h3> | ||
− | <p class="text-faded" style="font-size: 15px">Earlier this summer, we have discussed to explore different kind of biotech applications using synthetic biology. Apparently, many of these applications were limited by the availability of suitable chassis for the particular process involved. Therefore, the Edinburgh Overgraduate iGEM team decided to expand the availability of chassis for applications which needs specialised host organism: (1) Sustainable production of biomaterials through photosynthesis, (2) degradation of various pollutants/toxic wastes, and (3) robust production of secondary metabolites. | + | <p class="text-faded" style="font-size: 15px">Earlier this summer, we have discussed to explore different kind of biotech applications using synthetic biology. Apparently, many of these applications were limited by the availability of suitable chassis for the particular process involved. Therefore, the Edinburgh Overgraduate iGEM team decided to expand the availability of chassis for applications which needs specialised host organism: (1) Sustainable production of biomaterials through photosynthesis, (2) degradation of various pollutants/toxic wastes, and (3) robust production of secondary metabolites. |
− | + | <br><mark>Thus, we explore suitable organisms for the tasks and develop the means to do genetic manipulation and create a collection of necessary parts library.</mark> <br>In the process, we realized that by domesticating new organisms as chassis, we open up new risks and safety challenges towards users and environment. | |
+ | <br><mark>Therefore, we also developed a tool (software) to screen for potential risk from the organism’s database of toxic secondary metabolites.</mark> | ||
+ | </p> | ||
+ | <center><a href="https://static.igem.org/mediawiki/2016/0/08/T--Edinburgh_OG--Tom_Essay_WhyNeedMoreChassis.pdf" class="page-scroll btn btn-default btn-xl sr-button">An Essay on Expanding Chassis</a> | ||
+ | </center> | ||
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<p><b>Rhodococcus jostii</b> is a gram positive bacteria with extensive catabolic pathway to degrade variety of chlorinated compounds, such as polychlorinated biphenyls (PCBs). They are potential chassis for synthetic biology applications in bioremediation.</p> | <p><b>Rhodococcus jostii</b> is a gram positive bacteria with extensive catabolic pathway to degrade variety of chlorinated compounds, such as polychlorinated biphenyls (PCBs). They are potential chassis for synthetic biology applications in bioremediation.</p> | ||
<p><b>Penicillium roqueforti</b> is a filamentous fungi used in the production of blue cheese. They are biotechnologically relevant for the industrial production of enzymes (cellulases, pectinases, lipases, proteases and amylases) and could be used to produce complex secondary metabolites.</p> | <p><b>Penicillium roqueforti</b> is a filamentous fungi used in the production of blue cheese. They are biotechnologically relevant for the industrial production of enzymes (cellulases, pectinases, lipases, proteases and amylases) and could be used to produce complex secondary metabolites.</p> | ||
− | </div> | + | <center><a href="https://2016.igem.org/Team:Edinburgh_OG/Experiments" class="page-scroll btn btn-default btn-xl sr-button">Find out more!</a> |
+ | </center> | ||
+ | </div> | ||
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− | < | + | <h3>Golden Gate MoClo<h3> |
− | + | <p class="text-faded" style="font-size: 15px">Central to synthetic biology is the use of systematic assembly and sharing of parts between community members. As working in new chassis will emphasizes on characterisation of suitable parts and trial-error approach, we chose to adopt Golden Gate MoClo as our assembly standard. This approach enables the assembly of multiple parts and transcriptional units by parallel approach via BpiI (BbsI) in different levels (Engler et al., 2008; Weber et al., 2011). | |
+ | <br><br>To utilise MoClo, a set of destination vectors need to be developed to accommodate assembly in different levels. Unfortunately, the chassis we were working on (R. jostii) cannot utilise the origin of replication from available destination vectors, which are designed for Eschericia coli. Therefore, we developed a set of MoClo destination vectors for assembly and transformation to R. jostii. We have shown that our destination vectors can be used for combinatorial assembly and transformed to the host organism. | ||
+ | </p> | ||
+ | <center> | ||
+ | <a href="https://2016.igem.org/Team:Edinburgh_OG/Description#design" class="page-scroll btn btn-default btn-xl sr-button">Vector Design</a> | ||
+ | <a href="https://2016.igem.org/Team:Edinburgh_OG/Proof" class="page-scroll btn btn-default btn-xl sr-button">Proof of Concept</a> | ||
+ | </center> | ||
+ | </div> | ||
<div class="col-sm-4"> | <div class="col-sm-4"> | ||
− | <h3> | + | <h3>Developed Parts (Phytobricks)<h3> |
− | <p class="text-faded" style="font-size: 15px"> | + | <p class="text-faded" style="font-size: 15px">To utilise synthetic biology, a working parts (promoters, RBS, coding sequences) need to be developed as not all of them works orthogonally. We have developed a set of useful parts for expression in our chassis. |
− | + | </p> | |
− | + | <center><a href="https://2016.igem.org/Team:Edinburgh_OG/Parts" class="page-scroll btn btn-default btn-xl sr-button">Find out more!</a> | |
+ | </center></div> | ||
<div class="col-sm-4"> | <div class="col-sm-4"> | ||
− | <h3> | + | <h3>Human Practices<h3> |
− | <p class="text-faded" style="font-size: 15px"> | + | <p class="text-faded" style="font-size: 15px">The domestication of non-model organisms raises both biosafety and biosecurity challenges (e.g. unknown pathogenicity and toxicity). Such issues, if are not properly addressed, can be a risk for both users and their surroundings. The Edinburgh OG team worked to develop an accessible, easy-to-use program to evaluate the toxicity of curated secondary metabolites from organisms used as chassis. We have incorporate this aspect in our project and communicate with other teams in order to give contribution towards current risk assessment procedures and as precautionary step for both experienced and non-experienced users. |
− | + | </p> | |
+ | <center><a href="https://2016.igem.org/Team:Edinburgh_OG/Description#design" class="page-scroll btn btn-default btn-xl sr-button">Find out more!</a> | ||
+ | </center></div> | ||
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<p> 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. </p> | <p> 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. </p> | ||
+ | </div> | ||
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+ | <section id="design"> | ||
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+ | <div class="col-sm-8" style="background-color:white"> | ||
+ | <h2> Design of the Golden Gate Destination Vectors for Transformation in R. jostii </h2> | ||
+ | <p> We designed a set of Golden Gate destination vectors based on the MoClo standard to enable quick and easy way to create constructs and transformed it to our new Chassis, Rhodococcus jostii RHA1. We chose to use pSRKBB vector (Schmidt-dannert, 2014), which was derived from pSRK21 (Vesely et al., 2003) as the base of these destination vectors. This plasmid contains Corynebacterium replicon (pSR1) and has been successfully transformed to R. erythropolis, a closely related species of R. jostii (Knoppová et al., 2007; Vesely et al., 2003; Archer & Sinskey, 1993). It has also been refined by the removal of illegal sites incompatible with BioBrick standard (Schmidt-dannert, 2014). We removed the illegal sites (BsaI and BbsI) and replace the cloning site of the plasmid with a standard BioBrick cloning site flanked by two verification primer binding site (VF2 and VR), enabling colony PCR for quick screening of insert, derived from pSB1C3. The resulting vector (pSRKM_Empty) can be used as the template to make Level-1 destination vectors by digestion and ligation of standardized LacZα reporter flanked by fusion sites as described in Iverson et al. (2016). | ||
+ | </p> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/56/T--Edinburgh_OG--Matin_Design_pSRKM_vectors.png" height="783" width="700"> | ||
+ | <figcaption>Fig1. (A) Physical map of pSRKBB-Empty showing pSR1 replicon as a black band. The replicon contains per (a positive effector of replication) and rep (replicase) gene. pK19 backbone (white band) contains pBR322 ORI and Kanamycin resistance gene (KmR) regulated by the P45 promoter. Mutagenic primer binding sites are shown as red arrows (mut1, mut2, mut3, and mut4). The region in the red box was replaced by (B) a BioBrick cloning site flanked by standard verification primer binding sites, VF2 and VR, derived from Iverson et al. (2016), creating pSRKM_Empty. Destination vectors can be made by digestion and ligation with SpeI to insert standardized LacZα flanked by the designated Type IIs restriction enzyme (recognition sites shown as bold) and four base overhangs for Level-1 (C) or Level-2 (D) assembly. (E) Schematic map of the level-1 destination vector pSRKM_NN and (F) the level-2 destination vector pSRCM_NN with chloramphenicol resistance (CmR). X refers to the fusion sites and NNNN to its four base overhangs: A (GGAG), B (TACT), C (AATG), D (AGGT), E (GCTT), F (CGCT), G (TGCC), and H (ACTA).</figcaption> | ||
+ | </figure> | ||
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<h5>What should this page contain?</h5> | <h5>What should this page contain?</h5> | ||
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+ | <h2> References </h2> | ||
+ | <br> | ||
+ | <div class="newspaper" > | ||
+ | <p>[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 </p> | ||
+ | <!--<p>[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 </p> | ||
+ | <p>[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</p> | ||
+ | <p>[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 </p> | ||
+ | <p>[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. </p> | ||
+ | <p>[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 </p> | ||
+ | <p>[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. </p> | ||
+ | --><p>[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 </p> | ||
+ | <p>[9] Marles-Wright, J. (2016). Better by Design, Safer through Practice. Synbio LEAP Strategic Action Plan, (January), 1–5. </p> | ||
+ | <!--<p>[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 </p> | ||
+ | <p>[11] Demirbas, A. (2009). Biofuels. Securing the Planet’s Future Energy Needs. </p> | ||
+ | <p>[12] Dragone, G., Fernandes, B., Vicente, A. A., & Teixeira, J. A. (2010). Third generation biofuels from microalgae, 1355–1366. </p> | ||
+ | <p>[13] Spaeth, K. (2011). Polychlorinated Biphenyls, 126(4), 217–218. http://doi.org/10.17226/19865 </p> | ||
+ | <p>[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 </p> | ||
+ | --><p> Archer, J. A. & Sinskey, A. J. 1993. The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae. J Gen Microbiol, 139, 1753-9. </p> | ||
+ | |||
+ | <p> Iverson, S. V., Haddock, T. L., Beal, J. & Densmore, D. M. 2016. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synthetic Biology, 5, 99-103. </p> | ||
+ | |||
+ | <p> Knoppová, M., Phensaijai, M., Veselý, M., Zemanová, M., Nešvera, J. & Pátek, M. 2007. Plasmid Vectors for Testing In Vivo Promoter Activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Current Microbiology, 55, 234-239. </p> | ||
+ | |||
+ | <p> Schmidt-dannert, C. 2014. pSRKBB-empty (Plasmid #59449). www.addgene.org. </p> | ||
+ | |||
+ | <p> Vesely, M., Patek, M., Nesvera, J., Cejkova, A., Masak, J. & Jirku, V. 2003. Host-vector system for phenol-degrading | ||
+ | Rhodococcus erythropolis based on Corynebacterium plasmids. Appl Microbiol Biotechnol, 61, 523-7. </p> | ||
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Latest revision as of 19:52, 19 October 2016
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