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Contents 1 Safety First 1.1 List of organisms: 1.2 List of Hazardous Materials: 1.3 Safety issues in terms of research, environment and public safety 1.3.1 Bacterial Strains: 1.3.2 Bacteriophage lambda: 1.3.3 Biobricks: 1.3.4 Disposal: 1.4 Techniques and Materials 1.4.1 Ethidium Bromide: 1.4.2 SDS and Chloroform: 1.4.3 UV light: 1.5 Technion Safety Office 1.6 Misuse of the project 1.7 Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? 1.8 How could parts, devices and systems be made even safer through biosafety engineering?

Safety First

Biological hazard, also known as a biohazard, is an organism or a by-product from an organism that is harmful or potentially harmful to other living things, primarily human beings. There are four levels of biohazards, classified by the Center for Disease Control and Prevention (CDC) in the United States. A level 1 biological hazard poses the least risk while a level 4 poses the greatest.

List of organisms:

1. Escherichia coli, Top10
2. Escherichia coli, DH5α
3. Bacteriophage lambda

List of Hazardous Materials:

1. Ethidium Bromide
2. Sodium Dodecyl Sulfate (SDS)
3. Chloroform

Safety issues in terms of research, environment and public safety

All of the team members participated in lab and safety training given to us by our lab managers. They showed us how to use all of the instruments and materials in the lab in a way that wouldn't endanger us or the environment.

Bacterial Strains:

Since we are using standard E.coli strains (TOP10, DH5α) which pose no risk to either individuals or to the environment the vector strains used in our project are all level 1 biohazard. Therefore, when considering the "hazard factor" in the Risk = probability x hazard equation, our project wouldn't raise any safety issues to the researchers, the environment or to public safety, even in the case that the bacterial strains are released outside the lab. As for probability, we are taking the measures that are needed for level 1 biohazard, such as gloves, closed containers, and lab hygiene, to minimize the chance of the bacteria getting out of the lab.

Bacteriophage lambda:

Current viral gene transfer vectors are based on animal viruses that have significant drawbacks for clinical use, including potential safety. Bacteriophage lambda represents a new class of vector that has a long history of safe human use with minimal safety concerns because it is a bacterial virus without the capability to productively infect mammalian cells, therefore the probability and hazard of an infection are both minimal.

Since Bacteriophage lambda infects E.coli strains, working with this kind of organism requires special care and safety measures in order for the phage not to contaminate the E.coli strains in our department. Some of the research labs in our department are using E.coli strains in their research, hence, contamination with phage lambda might compromise their work. Moreover, humans have E.coli in their digestive system, therefore an infection of the phage might cause problems.

In order to deal with these safety issues we are taking the following steps: First, we work in a separate student lab free of bacterial strains that are used for research. Second, the first phase of working with the phage consists of the phage DNA alone, and not the phage as a whole; since the DNA cannot cause harm on its own it lowers the chance of infection during our work. Third, any work with the phage is carried out in a designated bench area with a set of dedicated instruments. Finally, additional safety measures are taken, such as: protective gloves, disposable coats, disinfection of the bench area with ethanol before and after usage, and a separate container for storage.

In conclusion, the biohazard risk of the phage to the researchers and the public is minimal, though the hazard to other bacterial strains in the faculty is higher. As described, we are taking precautions to lower the probability of the phage infecting bacteria in our faculty.

Biobricks:

None of the new BioBrick parts which we are making is known to have the capacity to confer pathogenic or toxic character onto E.coli or Bacteriophage λ.

Disposal:

As mentioned, there are four levels of biohazard (1-4). Level 1 consist mainly of bacteria and other microorganisms, which pose little risk in the case of exposure. Since our organisms are level 1, they can generally be disposed of in their own separate biological trash container. The same applies to disposable materials that came to contact with them, such as tips and eppendorfs.

Techniques and Materials

Most of the lab work involved no materials that demanded additional protection other than nitrile gloves and a lab coat.

Ethidium Bromide:

We regularly handle ethidium bromide when preparing agarose gels for separation and imaging of DNA fragments. According to the MSDS, ethidium bromide is highly hazardous in case of skin contact and fatal in case of inhalation, granting it level 2 Acute toxicity inhalation and level 4 Acute toxicity oral. To avoid exposure, there is a designated bench area and set of instruments (pipettes, tips) that are only used in protocols involving ethidium bromide. When working in this area or using equipment that has come into contact with ethidium bromide, exposure is avoided through the use of two sets of nitrile gloves. Moreover, Ethidium bromide is stored in a sealed container in a cool, dry, well ventilated location. After usage, the gels are thrown in a chemical waste bin for appropriate disposal.

SDS and Chloroform:

Both the SDS and Chloroform are used in the Alkaline Phosphatase Assay to detect translation of the pHO. Both has a certain biohazard when contact with the skin, the eyes or in case of ingestion. Chloroform also has carcinogenic and mutagenic effects, granting it level 2 in health hazard. As with the ethidium bromide, both have a separate working area with their designated equipment and ventilator. In case of Chloroform, eye protection is also required.

UV light:

Exposure to UV light is also something to be avoided while performing gel extraction. Therefore, to protect ourselves, we have been using appropriate shielding from UV lamps such as face shields, safety glasses, and whole body coats.

Technion Safety Office

We contacted the Technion safety office concerning our project. They saw no problems with our handling of biological safety since we are working with Level 1 organisms.

Misuse of the project

Our project is to create bacteriophage lambda, which attacks and causes lysis to specific cells only. Because we are not using a virus that can attack eukaryotic cells, the danger of misuse is minimal. If the project will be taken a few steps forward, as our vision dictates, and will be implemented in a virus instead of a bacteriophage, there may be certain risks.

Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?

Our idea is that a safety video will be created by the iGEM HQ and will be viewed by all iGEM participants. This will create a basic standard for safe research in synthetic biology. It is well noted that a video can't cover all of the safety measures needed to be taken into account while working in a lab. Yet, it will create awareness for safe lab work concerning not only researcher safety, but also environmental and public safety as well.

How could parts, devices and systems be made even safer through biosafety engineering?

As a preliminary solution, a failsafe mechanism, we believe that all strains that are used in the lab should be auxotroph for one or several organic compounds that aren't available outside the lab; this will lower its chances of survival.

An additional solution might be in the opposite direction: adding a lethal inductive gene, that in the presence of a certain chemical will cause the bacteria to die. This approach might be useful in the case of a known area or population that has been contaminated.

A more advanced solution, and far more complex, comes from the field of Xenobiology. In terms of safety, the creation of organisms that are invisible to natural biological forms, in the words of Markus Schmidt, the Organization for International Dialogue and Conflict Management, will allow us to reach a new standard of biosafety. Such organisms can be used without the risk of genetic and metabolic interactions with natural organisms, thus eliminating the risk of influence that genetically modified organisms pose on the natural world.