Our team, iGEM Thessaly, believes that safety is an essential part of science. It provides a robust definition of what is moral and ethical, and it preserves vulnerable communities as well as the environment. As a result, both in- and outside-of-lab safety considerations were thoroughly examined.

Laboratory safety, including the correct use of equipment and the avoidance of potentially hazardous incidents, is a top priority for us. During our research, we got significant advice and supervision about the use of laboratory equipment (such as autoclaves and laminar flow hoods) from our instructors, advisors and technicians to ensure that accidents will be promptly avoided.

Figure 1 & 2. Our laboratory facilities

Escherichia coli

We chose E. coli as a chassis organism to create the genetic constructs for our project. The strain of E. coli we used (DH5 alpha) during the project is non-virulent and therefore poses no immediate risk to humans or the environment. Although E. coli may cause a variety of diseases as an opportunistic pathogen, we took precautions to prevent contact with it by wearing lab coats, goggles and gloves when working with it, and by ensuring thorough hand always washing and hygiene.

Agrobacterium tumefaciens

For the purpose of transforming Nicotiana benthamiana, we also used Agrobacterium (strain GV3101) as a chassis organism. Similar to E. coli, its infection can lead to illness; however, we avoided coming into contact with it and spreading it by using lab coats, goggles and gloves when working with it and by maintaining strict hygiene standards.

Tobacco Plants (Nicotiana benthamiana WT)

Throughout our project we used Nicotiana benthamiana WT as the expression system for our genetic constructs. This required infiltrating them with genetically modified Agrobacterium tumefaciens in order to make transgenic plants. Even after creating transgenic plants, the plants themselves pose no harm to the environment or to humans. However, the plants were always contained within the laboratory and discarded properly when no longer used.

Some experiments required the use of hazardous substances or techniques. As instructed in the safety training, members of the team made sure to follow all the rules and guidelines to avoid causing any harm to themselves or the environment.

• Antibiotics were used for transformants selection. At high doses, they can be toxic when inhaled, ingested, or in contact with the eyes. Antibiotics were handled with appropriate safety measures and disposed of according to the disposal protocols.
• Ethidium Bromide was needed for the agarose gel visualization. As this chemical is a potent mutagen and is moderately toxic after acute exposure, team members followed all requirements to stay safe.
• UV light was used to visualize agarose gels with a gel imaging system. All safety measures were taken to avoid direct exposure to UV light.

Biosafety played a decisive role in the formulation and design of our project. In particular, the application of wetlands to water bodies was most demanding. The outcrossing of the GM plant is a major concern. Because the plants we would like to use are genetically modified, they should be properly contained and the whole system must be implemented with great care. Phragmites australis plants follow two main reproductive strategies: via seed dispersal and rhizomes. Therefore, we should consider two strategies: one for seed pollination and another to protect root dispersal. To this end, we intend on using a biosafety module to prevent pollination, mechanical barriers to enclose the plant's anthers and roots, and a temporal restriction to limit the platform’s implementation.

Seed Pollination

The sexual reproduction through seed dispersal is rare when the plant is placed on a wetland1. However, to ensure that our plant does not spread through pollination, we would need to employ the components BBa_K1554004 and BBa_K1554005, which include the coding sequence for the barnase protein and a tissue-specific promoter for the anthers, resulting in male sterility and the inability to produce seeds. To increase the protection during the application of the wetlands, the engineering department thought of some possible ways of mechanical barriers that address the above biosecurity issues. More specifically, for the upper part of the wetlands and the restriction of the anthers, we decided to build a structure that resembles the ecological character of our project. This structure incorporates the placement of native reeds around the structure for support and a cellulose mesh which will enclose the flowering part of the plant. As a final precaution, we will restrict the time frame during which the platform may be implemented to the time beginning in early April and ending in late July. This is because the blooming and cross-pollination period of the P. australis plants begins in early August and extends through the fall. In this way, we can prevent the spread of seeds.

Root Dispersal

The most significant issue we encountered was root system limitation, because rhizome fragments are the primary means of spread of common reed. Rhizomes can be broken apart by environmental conditions including wave and wind action, or by mechanical disturbance. After doing research, we discovered that there is a shortage of kill switch modules for plant roots and developing another one is an independent project. Following the advice of dr. Ralf Wilhem, a specialist in Biosafety in Plant Biotechnology, our dry lab department decided to focus on a mechanical solution similar to that of the upper part of the wetland. However, there is a fundamental difference in extra protection. Specifically, a protective mesh made from 100% upcycled PET bottles that stand out thanks to weather resistance, UV resistance, and durability. The above characteristics were necessary so that the mesh would not tear easily and would not corrode when applied to water. Finally, a water filter will be integrated on the mesh in order to limit the risk of parts of the root system escaping and therefore the risk of plant reproduction.

Figure 3. Protective Mesh and water Filter.

Root Kill-Switch

After iGEM, we will be able to consider and develop a novel synthetic circuit that activates the death of root cells in response to root breakage to fulfill the biosafety measures thoroughly.

Constructed Floating Wetlands Considerations

During the mechanical design of the wetlands, various alternatives were suggested for the protection and limitation of the root system, such as compartmentalization. We thought of the construction of two underwater tanks, in one of which the inflow of water would be allowed and would lead to the root system in which agroinfiltration would take place. The water is then transferred to a second tank where an outlet with a filter is located to ensure no part or cell would escape from the roots. However, this specific case could not find application due to the limitation caused by buoyancy.
Our future goal is to develop a better mechanical protection system that will be more ergonomic and efficient.

RC Boat Considerations

Constraints in the construction of the RC Boat were minimal and not of particular importance. As its construction materials are all biodegradable and environmentally friendly, the environmental footprint during its application is minimum. Nonetheless, in case of failure, we would not want its electronic and mechanical parts to end up at the bottom of the water system. For this reason, our next objective is the utilization of a protection system that will calculate the possibility of mechanical failure and give an order for a forced return of the RC Boat to the shores.