Safety

Safety introduction

The project with all of its involving laboratory work has been planned with a focus on safety from the start. Our principal investigators have explained the possible risks involved and educated us on the safety precautions of our lab. We have been given a walkthrough as well on laboratory equipment, emergency exits, emergency shower and further safety aspects. Our Primary PI: Prof. Per Hammarström is also a member of the biosafety committee of Linköping University and has held a lecture on lab safety.

Types of bacteria used

We have used Escherichia coli (E.coli) and Vibrio natriegens (V.natriegens) for our project.

E.coli strains:

- BL21 DE3 Gold
- NEB5 alpha strain (DH5alpha)
- Top10

V. natriegens strain:

-Vmax

Both E. coli (laboratory strains) and V. natriegens strains are class 1 (low-risk organisms) and is considered non-pathogenic. They should not survive outside of the laboratory, without proper media. But extra precautions were used when working with V. natriegens, the handling of it was in a strictly confined area and with designated equipment. They do not generally cause disease in healthy people, and have a low risk to cause environmental damage.

Laboratory safety

We received training in lab safety and risk assessment from our PI:s. This involved knowing the differences between different biosafety levels. Wearing appropriate safety equipment for different experiments using the correct intended facilities. Also using the correct microbial techniques when working in a lab, avoiding contamination, sterilizing all surfaces and involved equipment after use.

All team members also have at least 1,5 years of laboratory experience beforehand. Team members are not allowed to be alone in the lab or work after 23:00-05:00 and during weekends. Before each experiment a risk analysis was being made making sure all the necessary precautions were taken using the right safety equipment. We used an autoclave to sterilize our equipment and destroy all bacteria after use.

To handle potential risks with the novel peptides, we have taken measurements during the selection. The potential peptides that the model presented were filtered for cytotoxicity using two tools: BLAST which analyses if the peptide has a similar structure to a cytotoxic protein and ToxinPred an online cytotoxicity prediction tool (http://crdd.osdd.net/raghava/toxinpred). By BLASTing the peptide sequence we were able to get hints of the peptides functions. Peptide candidates were discarded if any sign of toxicity was present. Even though the approved peptides showed no sign of toxicity they were still handled with great precaution in fume cabinets

A potential risk can be associated with the novel peptides created from the modeling part of the project. Since these sequences are completely synthetic and have never been investigated prior to this. They were handled with care and considered a potential risk factor. However, the antimicrobial peptides predicted by the model has no known risk association.

The PI:s have reviewed the selection of the novel peptides created from the model by guiding us with what are important parameters to take into account when selecting the novel peptides. During this consulting, the risk aspect has been one major focus point.

We made sure of the correct usage of protective clothing (e.g lab coats, protective eyewear) when such was needed. Fume cabinets were used when handling volatile chemicals. Protective headgear, glasses, lab coats and gloves were used While working with the UV-table. We were also instructed in the use of other emergency tools such as: fire extinguisher, fire blanket and emergency showers. Special safety precautions will be applied depending on the experiment conducted. For example, if not handled properly during DNA staining, Ethidium bromide can pose a certain risk. Therefore Ethidium bromide was used only in a specially designated area with its own fume hood, using designated thicker gloves. Face masks are used to prevent inhalation of SDS.

All in all we have taken all the necessary precautions in order to minimize risks associated with our project, as stated above. We both regarded iGEMs rules and policies together with our university's rules and policies.