Safety form

Safety within the lab and the safety of our product outside of the lab are two factors that we as a team take very seriously. Working with modified biological organisms poses specific risks which must be addressed. Below we provide information about the safety training we have received and the safety protocols that we follow in the lab. Which host organisms we have used and the biological coding sequences utilized have also been provided. As scientists, we have a responsibility to our own safety as well as the safety of the public.

1. What safety training have your team received?

   Our team have received safety training regarding waste disposal, biosafety, fire safety, handling dangerous materials and chemicals, emergency exits, working with microorganisms and personal protection. Everything corresponding to a BSL 1.¹

2. Who is responsible for biological safety at your institution and what are the biosafety guidelines of your institution?

   We have had a meeting with those responsible for Biosafety at our institution (IFM – Institution for Biology, Chemistry and Physics at Linköping University) and they raised no issues with our project.²

3. The organisms and parts that you use

   See document: LinkopingUniversity_Safety2014_Spreadsheet.pdf

4. Does the lab work of your project pose any risks to the safety and health of team members or others working in the lab?

   1. In the project, we used Escherichia. coli BL21 DE3 strains (BL21 DE3 Codon + and BL21 DE3 supercompetent cells) which fall under Risk Group 1, corresponding to the certification of our laboratory (Biosafety Level 1). Before using the strains, the team has researched risks relating to usage of these strains. There are minimal risks associated with these strains.

   2. There are risks in indirect and direct bacterial contamination from handling E.coli strains without proper safety equipment. This can lead to sickness if not properly attended to (this risk is highest when the contaminated individual has an open wound/cut or ingests E.coli).

   3. There are risks involved when using bacterial strains that are transformed with antibiotic resistance (Amp and Chl). Contamination with antibiotic resistant bacteria can be dangerous for the individual. Furthermore, there are risks involved with transforming plasmids which code for phenotypes that the E.coli host does not naturally have, risk for horizontal gene transfer from GMOs to wild type bacterial strains.

   4. Enzymes used during restriction digests and proteases used when extracting DNA can cause allergenic reactions.

5. Does the lab work of your project pose any risks to the safety and health of the general public, if any biological materials escaped from your lab?

   1. There is a risk for bacterial infections (E.coli) if bacteria were to be let out in the water-ways and consumed by the public

   2. There are risks involved when using bacterial strains that are transformed with antibiotic resistance (Amp and Chl). Contamination with antibiotic resistant bacteria can be dangerous for the individual. Furthermore, there are risks involved with transforming plasmids which code for phenotypes that the E.coli host does not naturally have, risk for horizontal gene transfer from GMOs to wild type bacterial strains.Risks to the safety and health of the environment:

6. Does the lab work of your project pose any risks to the environment, from waste disposal, or from materials escaping from your lab?

   There are no hazards to the environment were identified for the following reasons:

   1. Incorrect handling of waste contaminated with bacteria. Our team could miss autoclaving material contaminated with bacteria, which would then end up in the environment. We have however not transformed anything into our bacterial strains to make them more competitive than bacteria in the native environment. Therefor we consider it highly unlikely that our modified bacteria could compete with natural strains in their native environments. Our product doesn´t have any environmental application, so if bacteria either escapes the lab or ends up in the environment due to incorrect handling of waste the risk of environmental contamination is very low. Another risk could be the use of antibiotics; however our team only uses degradable antibiotics therefor not being harmful to the environment or contributing to the rising problem with antibiotic resistance.

   2. Bacteria undergo a natural process called horizontal gene transfer, this process is when DNA is transferred from one bacterium to another. Bacteria are also subject to evolutionary forces, in which the fittest bacteria survive. Therefore, it is possible that escaped modified bacteria could cause unwanted and even dangerous effects in the environment. A few possible scenarios are the transfer of genes to native pathogenic bacteria that would give them a new evolutionary advantage, or released GMOs spreading in an ecosystem or even our bodies, overpowering populations of natural bacteria.

7. Does the lab work of your project pose any risks to security through malicious mis-use by individuals, groups or countries?

   The bacteria that we use are not a threat to security. However, if the plasmids contain genes that express harmful proteins then they could implicate a risk to security. Engineered bacteria that express harmful proteins could be used as bioweapons; however, our product doesn’t contain any proteins that pose a risk for security.

8. What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.)

   We received a safety education (BSL 1) regarding biosafety and disposal of contaminated waste which was conducted by the biosafety officer from IFM (institution of biology and chemistry). All material which has been in contact with bacteria is thrown in a specific waste followed by autoclaving. We always make sure that our modified bacteria are kept at restricted areas and that there is no risk of contamination outside the laboratory. When working with bacteria cells we always use sterile conditions and a flame at the bench being exposed of bacteria. Every surface and material being exposed of bacteria is sterilized. Gloves, lab-coat and in some cases goggles are always used when we work in the lab due to individual as well as group safety. We never leave lab when gloves are being used and the lab-coat is used to avoid contamination of our clothes. Furthermore, we have reduced the risk of unwanted protein production in the nature due to the dependency of an inducer for protein expression. Our used plasmid which is transformed into the bacteria´s depends on an inducer such as IPTG for protein expression which means that no protein production can occur at all as long as no inducer is added. Furthermore, the inserted sequences encode only short fragments of proteins and are highly unlikely to confer any harmful phenotype on the disabled E.coli host. Since our final product do not contain any living material such as bacteria (because they have been lysed prior to use) then there are no safety issues at all considering the health of people as well as the safety issues regarding researchers, the public or the environment.

9. Are there any risks of your project in the future?

   1. The risks of producing our two protein complex should not be higher than of production of recombinant proteins today in larger scale. Due to the fact that only pure protein will be available to the end customer they will never be in contact with bacteria. Sufficient protocol for purification must be determined and even if bacteria go through to the end formula, unless swallowed, it will not be any higher risk than previously described. Standard biotechnology procedures was used and will be used, resulting in no new knowledge or new methods generated. Therefore no new risks will develop. One risk might arise from the fact that the product becomes widespread and popular which could in turn lead to people with allergies becoming less cautious. They could begin to depend upon the detection system and stop bringing their Adrenaline shots. Another form of misuse would be to terrorize people allergic to peanut through mass production of the allergen.

   2. At the moment, if the protein sample aren’t fully purified from bacteria and someone swallow it, our project product is pretty much safe. In the future, to make it even safer would be to implement sealed disposable cuvette which the user can throw away.