Team:Edinburgh OG/HP/Gold

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GOLD REQUIREMENT

How the investigation of biosafety issues have been integrated
into the design and execution of the ExpandED project.



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This year’s Edinburgh OG iGEM team has worked on a program that allows the evaluation of curated secondary metabolites’ toxicity produced by a range of microorganisms (bacteria and fungi). The program is intended to complement the current risk assessment practices by constituting an additional precautionary step that is to be followed before any work in the laboratory with the reviewed host strain begins. Furthermore, it is intended to be accessible to a wide constituency of experts in the field, trainees, DIY biologists, other stakeholders and the general public.


This tool, named “CARE” - Chassis Assessment & Risk Evaluation
includes information about 30 microorganisms:


● The microorganisms from the Edinburgh OG 2016 iGEM team: the cyanobacterium Synechosistis sp. PCC 6803, the bacterium Rhodococcus jostii RHA1 and the fungus Penicillium roquefortii.

● The non-model organisms used by iGEM teams based in North America in 2014 and 2015 (as documented by the Yale 2015 iGEM team): Chlamydomonas reinhardtii, Chlorella kessleri, Chlorella vulgaris, Flavobacterium psychrophilum, Gilliamella apicola, Lactococcus lactis, Rhizobium tropici CIAT 899, Snodgrassella alvi and Synechococcus sp. PCC 7002.

● The industrially relevant Shewanella oneidensis, Saccharopolyspora erythraea, Myxococcus xanthus, Cupriavidus necator, Chromobacterium violaceum, Carnobacterium maltaromaticum, Sorangium cellulosum and Pseudomonas fluorescens.

● The model and non-model microorganisms with an annotated genome from a selection of this year’s iGEM teams (as seen in the Outreach section): E. coli BL21DE3, E. coli C41, E. coli MG1655, Yarrowia lipolytica, Saccharomyces cerevisiae W303, Dechloromonas aromatica, Pseudomonas putida KT2440, Synechococcus elongatus PCC 7942, Pichia pastoris, Myxococcus xanthus and Acinetobacter sp. ADP1.


Along with these microorganisms, a database with the related secondary metabolites information (i.e. the general class of secondary metabolite, the most similar biosynthetic gene cluster (BGC) related to the secondary metabolite, its location in the genome and the percentage of gene similarity for each annotated microorganism) was constructed using the Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) software.

It is worth mentioning that the databases will be able to be updated by integrating new microorganisms or editing existing ones simply by logging-in and waiting for the validation of the data by the program administrators.



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According to the toxic effect of these compounds on human health, animal health and the environment, a quantitative criterion for clear toxicity scores was created using a scale from 1 to 5 and a “traffic light” colour scheme. If the reviewed information showed that the secondary metabolite did not affect in a negative manner any of the three parameters, it was considered non-toxic and categorised as a “1” (green). If the data showed that the compound exhibited some degree of toxicity but nothing considerably serious or untreatable, it was considered slightly to moderately toxic and scored as a “3” (yellow). And if it showed that the secondary metabolite was extremely toxic, it was considered as highly toxic and classified as a “5” (red). However, considering there were three parameters to be contemplated, combinations of those three scores could occur (e.g., a secondary metabolite being considered non-toxic for humans but slightly to moderately toxic to animals). Therefore, if a compound was non-toxic according to one parameter and slightly to moderately toxic for the others, it was categorised as a “2” (green-yellow). On the other hand, if it was slightly to moderately toxic according to some parameter and highly toxic for others, it was classified as a “4” (yellow-red). Finally, if the secondary metabolite had no documented toxicity information, or if there was no specific BGC and just the general class of secondary metabolite or microorganism, the compound was categorised as “?” (black) with the note to consider if, according to the precautionary principle , one should prusue with the use of the given microorganism in the laboratory.

As a proof of concept, the 30 organisms were screened using the CARE tool and the results showed that, although some secondary metabolites were considered as toxic (e.g. roquefortine, PR-toxin, isofumigaclavine and mycophenolic acid for P. roqueforti), their amounts do not affect human health, meaning that these organisms were safe to work with in laboratory settings and we could proceed with the experimentation with them.

When a laboratory works on a non-model organism with the objective of harnessing its industrial potential, genome editing processes may be involved (e.g., CRISPR-Cas9 tool). Therefore, this tool could additionally be used in order to determine differences in toxicity between native and genetically modified organisms.