Difference between revisions of "Team:Edinburgh OG/Description"

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                     <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).
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                     <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.                    
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                      <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.  
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Revision as of 13:32, 19 October 2016

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Project Summary

Motivation

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. Thus, we explore suitable organisms for the tasks and develop the means to do genetic manipulation and create a collection of necessary parts library. In the process, we realized that by domesticating new organisms as chassis, we open up new risks and safety challenges towards users and environment. Therefore, we also developed a tool (software) to screen for potential risk from the organism’s database of toxic secondary metabolites.

Our Chassis

Synechocystis sp.

Rhodococcus jostii RHA1

Penicillium roqueforti

Synechocystis sp. is one of the most extensively studied cyanobacterial species, a unique class of microorganisms that are able to fix and metabolize carbon dioxide using the energy derived from sunlight. They are potential chassis for synthetic biology application in biomass production and carbon sequestration.

Rhodococcus jostii 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.

Penicillium roqueforti 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.

Golden Gate MoClo

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).

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.

Developed Parts (Phytobricks)

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.

Human Practices

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. Thus, we explore suitable organisms for the tasks and develop the means to do genetic manipulation and create a collection of necessary parts library. In the process, we realized that by domesticating new organisms as chassis, we open up new risks and safety challenges towards users and environment. Therefore, we also developed a tool (software) to screen for potential risk from the organism’s database of toxic secondary metabolites.

Project Description

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.

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.

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.

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