Safety

We took the requirements of lab work and project safety very seriously by carefully studying safety protocols and preparing our work surroundings, as well as incorporating a specific biosafety-directed element into our SynORI framework.

General laboratory safety

Prior to starting lab work, we analyzed the safety level of the laboratory and classified it as Biosafety level 1. Then we read and learnt to apply and utilize the safety protocols, which were ought to be carried out at this level of Biosafety. These protocols included:

• decontamination of materials prior to disposal, using an autoclave

• personal protective equipment (lab coats, eye protection, and gloves)

• mechanical pipetting only

• the prohibition of food, drinks, and any smokable materials, etc.

Furthermore, the whole lab team had previously worked in laboratories of the same biosafety level, hence they were experienced and familiar with the work requirements and environment. In addition, the team was introduced to all of the safety equipment locations and safety procedure routes specific to this particular laboratory space.

General laboratory safety protocols and precautions

• Specific safety equipment: the locations and operating procedures of emergency showers and eyewashes, as well as the fire extinguisher and the panic button

• Waste disposal: the team was introduced to autoclaving protocols and specific chemical waste disposal protocols according to the compounds used.

• Personal protective equipment: lab coats, eyewear, and enclosed footwear were used to minimize the possibility of skin contact area with harmful substances. Before leaving the lab, the team made sure to disinfect and sterilize their clothing to prevent the contamination of external environment .

• Chemical safety: the locations and appropriate storage methods for both flammable and potentially hazardous chemicals, as well as the required working environment protocols to handle these substances: working in an active fume hood, etc.

Application safety issues

The application of our project will have minimal required safety regulations as it will mostly be employed in the laboratory setting with strictly controlled environment.

Nevertheless, all of the GMO required safety precautions need to be taken into account. We emphasize that the antibiotic resistance gene from our SynORI system cannot be effectively transmitted to other cell species via horizontal gene transfer, because it is split into two coding regions located in two different vectors, which are also locked on a translational level. Furthermore, as we developed a multi-plasmid system consisting of 5 or possibly more plasmids, it would require the same amount of antibiotic resistant genes to externally select all of the plasmids, but due to our ingenuity we have reduced the amount to only one antibiotic resistant gene, which is split through all of the 5 plasmids.

Project specific laboratory safety

We also utilized some compounds that required additional safety protocols:

• Ethidium Bromide: This compound is a potent mutagen and is moderately toxic after an acute exposure. Therefore, the lab team took it upon themselves to familiarize with the appropriate procedures that are required to handle cases of spills or acute exposures. These procedures included working in an active fume hood and wearing personal protective equipment (PPE) to minimize the chance of exposure, as well as extensively studying the material safety data sheet (MSDS) for Ethidium Bromide. We disposed of the product in accord with the disposal protocols and the state safety regulations.

• Sybr green: the compound is known as a much safer replacement for the human mutagen Ethidium Bromide, due to its minimal carcinogenic effects when compared at equal concentrations with Ethidium Bromide itself as well as requiring a less complex mode of disposal. However, the team still took the necessary precautions to study the material safety data sheet (MSDS) for this compound, wore personal protective equipment (PPE), and disposed of the waste in accord with the protocol and the state regulations.

• Antibiotics: we used tetracycline as our main antibiotic for selection. According to the material safety data sheet (MSDS) for tetracycline, ampicillin, chloramphenicol, and kanamycin, these compounds are known to have hazardous effects at high concentrations and both acute and chronic exposure to humans via inhalation, ingestion, or contact with eyes. Therefore, the team wore personal protective equipment (PPE) when handling the compound, studied the safety protocols and were equipped with the necessary information to handle spills, acute exposures, and disposed of the product in accord with the disposal protocols as well as the state safety regulations.

• UV light: we used the UV lights both to sterilize our boxes plus equipment (including PIPETMAN pipettes, dishes) and to look and cut out gels. Our team members wore protective glasses, which fell under the personal protective equipment criteria. Due to very few personal close-up encounters with UV and taken safety precautions, we completely minimized the possible hazards of encountering UV light in the lab.