Laboratory procedures we have undertaken that have risks associated with them are as follows:

• Pseudomonas sp. will be transformed with GFP to prove transformation of said species is possible.

• Kill curves with multiple antibiotics, including chloramphenicol, will be produced for Pseudomonas sp.

• Several chemotaxis assays will also be conducted on the free-living nitrogen fixing bacteria (Azorhizobium caulinodans (ORS571), Azospirillum brasilense (SP245), Herbaspirillum seropedicae (Z67)) to observe their interaction with naringenin. Kill curves for naringenin will also be produced for all bacteria.

• Genes encoding enzymes of flavonoid biosynthesis will be introduced into E. coli to prove the system can function in bacteria before transferring it to Pseudomonas sp.(time permitting).

• As Pseudomonas sp. is a plant endophyte, its colonisation efficiency will be tested in Arabidopsis roots.

We have been working alongside academics at the University while developing our project to ensure we are working safely.

Dr. Matthew Peake is the Senior Biological Research Technician at Newcastle University and we have been working closely with him to ensure all lab protocols/safety measures are adhered to.

Additionally, we are working with Dr. Vasilios Andriotis who has extensive knowledge on seed biochemistry, especially with Arabidopsis species, and Dr. Maxim Kapralov who currently engaged in research around plant biology and photosynthesis. Dr Maria Del Carmen Montero-Calasanz also has prior experience working with Pseudomonas sp.

Aseptic technique was used to prevent biocontamination and unintended release of organisms> In addition, all waste was incinerated or autoclaved. The team used non-pathogenic strains of organisms where possible to mitigate the risks identified.

Hazards of Flavonoids

• Naringenin – skin, eye, respiratory irritation.

• Luteolin – potential skin and eye irritation

• Scutelarin – non-hazardous

To avoid any irritation or inhalation, all handling of the various flavonoids was undertaken in the fume hood. For the weighing of the flavonoids when they are in their powdered form, face masks were worn to minimise the risk of inhalation. The area surrounding was kept vacant to avoid others not wearing masks coming into contact with the powder.

Safety specs were worn to avoid eye irritation, as well as gloves and lab coats to avoid skin irritation.

Biological Hazards

• Escherichia coli (DH5α)

• Herbaspirillum seropedicae (Z67)

• Azorhizobium Caulinodans (ORS571)

• Azospirillum brasilense (SP245)

These are low risk; however, they may have the potential to cause low level, localised changes to soil nitrogen content.

Pseudomonas sp. (CT 364)

Pseudomonas sp. strains can be opportunistic pathogens after repeated exposure resulting in infections of the mouth, stomach and lungs; however, the species is less virulent than others in the genus. Very low risk of infection unless immunosuppressed (e.g. UTI), potential to colonise native plant species if released. Not listed as human pathogen.

Experiments are performed in a restricted lab; lab coats and gloves are worn to reduce risk and hands are washed afterwards.

Growing in Contained Environments

The use of biotechnology to modify plants has become common place in agricultural research - but we have conceptualised this as common place in agricultural practice. (See our Human Practices). In the context of our project, the purpose of containment is to prevent recombinant DNA from transgenic organisms being transferred to populations outside of our urban farm in Newcastle’s Victoria Tunnel (although the safety principals apply to any contained environments in which our transgenic organisms are present).

Genetically engineered organisms are subject to special rules intended to ensure that they are used in a way that does not pose an unacceptable risk to human health - or the environment. In order to design our hydroponic urban farm to meet these biosafety standards, we referred to:

• Greenhouse Research with Transgenic Plants and Microbes: A Practical Guide to Containment
  (Traynor, Patricia L, Dann Adair, Ruth Irwin)
  Methods for the safe handling of transgenic materials in contained environment are also described in the National Institutes of Health’s Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines).

Below I have taken extracts from the guide to containment that we referred to in order to design our own contained system:

• Elements of containment:
  1. Avoid unintentional transmission of rDNA-containing plant genomes or release of rDNA-derived organisms associated with plants;
  2. Minimise the possibility of unanticipated deleterious effects on organisms and ecosystems outside of the experimental facility;
  3. Avoid the inadvertent spread of a serious pathogen from a greenhouse to a local agricultural crop;
  4. Avoid the unintentional introduction and establishment of an organism in a new ecosystem.

Having read the guide, we concluded our GMO was classified as BL2-P.

• Biosafety Level 2 for Plants (BL2-P)
  "BL2-P is assigned to experiments with transgenic plants and associated organisms, which, if released outside the greenhouse, could be viable in the surrounding environment but would have a negligible impact or could be readily managed. BL2-P is required for transgenic plants that may exhibit a new weedy characteristic or that may be capable of interbreeding with weeds or related species growing in the vicinity."
• Procedures that must be followed for BL2-P:

Newcastle's Victoria Tunnel - Retrofitting for Containment

Retrofitting a structurally sound facility to meet BL2-P containment standards is far cheaper than building a new facility. Necessary modifications, if any, are usually simple, straightforward, and involve readily available materials. This is one of the reasons we have proposed a contained environment in the Victoria Tunnel - it is in a prime location running under the city centre whilst being structurally sound and accessible.

Physical containment is achieved through making appropriate choices when it comes to facility design and equipment. These choices include: glazing, sealing, screening, air flow system, and other features all affect the degree to which a contained environment is capable of isolating transgenic organisms from the surrounding environment. These systems are also effective in keeping unwanted pests out of the facility.

Layout

When retrofitting to accommodate transgenic materials: traffic patterns, process flow, and security measures should be analysed to determine if the layout should be modified. The configuration should be optimised to provide variable levels of containment and growing conditions, control of access, and ease of movement.

An efficient and manageable layout has an array of small rooms and cubicles opening off one or more common walkways; a compartmentalised arrangement of small rooms allows the facility to provide a variety of containment levels as well as individualised environmental conditions.

A contained environment can be an inhospitable for people and equipment because of the humidity, temperature, light, chemicals, and soil. An enclosed area within or adjacent to the facility, provides cleaner, more comfortable space for offices, labs, equipment, supplies, and control systems.

Additional safety considerations when designing contained environments

• Termination and Disposal
  "To prevent the unintended survival of GMOs outside the contained environment, all experimental materials must be rendered biologically inactive (devitalised) before disposal. Termination procedures for the safe disposal of soil and plant material should be part of the experimental plan for a research project. Devitalisation of plant material and soil should be completed before it leaves a contained facility to go to landfill."
• Apparel and Hygiene
  "Personnel entering BL1-P and BL2-P facilities may wear their usual street or lab clothing."


• Greenhouse Staff
  "All staff should become familiar with any differences between caring for GMOs and conventional plants that may affect their own work. In most cases, a brief orientation session is sufficient to explain the nature of the plants (or other transgenic organisms) and any special practices to be employed when handling or working around them. Both the greenhouse manager and the PI should work with the staff to ensure compliance with safety procedures and standards."
• Signage
  "Entryways into BL2-P and higher facilities should be posted with signs indicating that access is limited to authorised personnel only. If the facility uses organisms that pose a risk to the local ecosystem or agriculture, a sign so stating must be placed on the access doors to the facility. A description of the potential risk may be posted on the restricted access sign as long as this is not confidential information. The sign should state the name and telephone number of the responsible individual, the plants in use, and any special requirements for using the area. It may include contact information for the greenhouse manager and others to be called in case of emergency."