Team:Macquarie Australia/WetLab/Safety

Safety

Safe and responsible laboratory procedures are required to ensure the continued efficient operation of research, teaching and experimental laboratories. In preparation to undergo our iGEM project, we considered the risks of working in the lab and the steps that would be taken to mitigate them, in view of the best practice of the domestic and international scientific communities.

Introduction

As undergraduate students nearing the end of their degrees, the whole team was familiar with proper lab safety and procedure. We held a briefing again at the commencement of the project to familiarize ourselves with biosafety protocols in consideration of the materials and methods that we would use. The team was briefed on biosafety from university safety officers, including technical staff. This has involved a review of lab safety including chemical safety (MSDS’s, risk assessment) and the gene modification act. We have received specific instruction as to the safe and efficient operation of the laboratory equipment and training processes. The induction also involved a review of proper personnel protective equipment (PPE).

Australian Lab Safety Protocols & Macquarie University

• Biosafety at Macquarie Institutional Biosafety Committee
• Biosafety at Macquarie University Faculty of Science
• Chemical Safety at Macquarie University
• Gene technology act

The Biosafety committee of Macquarie University is responsible for overseeing biological safety. Their involvement with the Macquarie University iGem project in discussion has been primarily to ensure that the project complies with their guidelines. We have been given approval from our Biosafety committee for our project. They had no concerns in relation to our project provided that the listed biosafety guidelines are followed.

The Australian Government's legislative guidelines regulate Australian biosafety, and the Health Department's Office of the Gene Regulator is the responsible regulator for these guidelines. The Office provides a full list of relevant Australian legislation which may be found at this link. We have therefore met the standards and requirements set by our internal safety committee, domestic requirements and international guidelines

Organisms and Parts

Risks of our Project

The main risks of our project arise from contact with micro-organisms (in this case,  E. coli ), which may cause irritation to skin or eyes in susceptible individuals, and is chemically harmful if ingested. The ability of E. coli K-12 strains, as being used, to colonise and express toxicity [1] have been entirely mitigated by an inability to form biofilms[2]. They are of minimal risk to humans, expressing little of the pathogenesis of wild  E. coli  strains [3].

These strains pose very minor health risks the to community; a slight risk to immuno-compromised individuals that accompanies most microorganisms will also arise for our organism.

These strains, due to the nutrient and storage chemicals used, are potentially harmful to aquatic organisms via waste release. Preservation and nutrient factors (such as glycerol ) pose specific risks to crustaceans, aquatic plants and algae, including Daphnia Magna , Oncorhynchus mykiss.

To the best of our knowledge, there is no potential for the misuse of our material from either the bacterial expression host or the genes utilised in altering the function of the host. As the host is a K 12 strain derivative, it possesses no comparative potential as an bioagent over natural types of E. coli, possessing less virulence and pathogenicity as an infectious agent in humans, and little in animal species [4]. Consequently, we do not consider our material to pose a risk to security as it is not a suitable bacterial expression host for the creation of bioweapons in this respect.

Secondly, our operons do not pose a risk to security through misuse. They are involved in the conversion process of protoporphyrin to chlorophyll a, which does not produce a reactive compound or dangerous product. This pathway, when inserted alone, is fatal to the host in sunlight, as photosystem II is incomplete, as no reaction centre chlorophyll-protein complex is present to disperse charge. As these operons would be fatal to another non-photosynthetic host without the genetic insertion for the reminder of the photosynthetic pathway, we do not anticipate our project poses a risk to security.

Risk Reduction Strategy

The Macquarie team has therefore taken measures to reduce these risks:

• Human Safety: Preventative measures, namely, safety gear (PPE) and training, ensure that contact with chemicals, bacteria and genetic materials is avoided. The laboratory is equipped with the appropriate Biosafety Level 1 materials and is in adherence with the code of practices proposed by WHO.
• Ecological Safety: As flagged, the direct disposal to waterways of our biological materials is not acceptable: they pose a direct biological hazard to organisms and ecosystems if released into the environment. Autoclaving and landfill ensure that waste is disposed of in accordance with Australian and WHO guidelines for this type of waste, and is consistent with our own risk consideration.
• Material Control Staff and team members are not permitted to utilise the lab out of hours; nor remove materials from the lab. Lab coats ensure that material is not accidentally transferred outside of the laboratory by spills or contamination, again consistent with WHO and Australian guidelines. Additional measures (the counting and control of materials) are not necessary.

Risks into the Future

Our project provides a biotechnology option to bridge solar energy to combustion engines. Moreover, acts as a carbon sink by which carbon can be sequestered from the atmosphere or from carbon intensive processes. It is possible that industrial production through many life cycles of the organism may lead to evolutionary pressure in E. coli, toward producing undesirable products or the expression of virulence factors. This may be addressed by preventing contamination or escape of the organism to prevent wild-type plasmid transfer into the colonies and regularly assessing the genetic composition of the industrial  E. coli.

The knowledge of how to insert a pathway to store sunlight in a reactive substrate poses little inherent risk. However, we have been considering that our research may harness artificial photosynthesis by significant modifications of the P700 photosynthetic pathway to produce hydrogen gas instead of sugar [5] [6]. Hydrogen is flammable and the risk will accompany production and storage. It will be necessary to properly contain and store the organism and it's products away from sources of ignition and to protect it from excessive heat.

Antibiotic resistance genes are used to mark the operons of genetically engineered industrial organisms. It is unlikely that these genes will be able to transfer antibiotic resistance to wild-types and therefore cause issues. Despite the minimal hazard that it poses, the careful control of material and destruction of materials for environmental purposes fulfils much of the same effect.

As the project currently does not include a complete photosystem II and possesses no photosystem I, it has a built in control mechanism. The cells cannot presently survive exposure to sunlight (unguided electron resonance destroys the cell). We discussed whether a 'kill switch' (an operon designed to induce cell death upon the introduction of a chemical signal) would be appropriate for this organism. We decided against it for two reasons: the cell was too fragile to survive exposure to light, and the extremely low risk of the project did not justify engineering additional fragility into the organism.

References

• Escherichia Coli K-12 Derivatives Final Risk Assessment | Biotechnology Program Under Toxic Substances Control Act (TSCA) | US EPA .
• O Vidal et al, ‘Isolation of an Escherichia Coli K-12 Mutant Strain Able to Form Biofilms on Inert Surfaces: Involvement of a New ompR Allele That Increases Curli Expression’ (1998) 180 Journal of Bacteriology2442.
• CA Fux et al, ‘Can Laboratory Reference Strains Mirror “Real-World” Pathogenesis?’ (2005) 13 Trends in Microbiology 58.
• Escherichia Coli K-12 Derivatives Final Risk Assessment | Biotechnology Program Under Toxic Substances Control Act (TSCA) | US EPA, above n 1.
• Daniel Hallén and Robert Rundberg, ‘Mathematical Modelling of Natural and Artificial Photosynthesis’.
• Ping Huang, EPR Studies of Ruthenium-Manganese Complexes as Biomimetic Models for Photosystem II - Approaching Artificial Photosynthesis (dissertation, Lund University, 2003) .