Biosafety

1. Biosafety procedures in the lab - What for?

   As the World Health Organization (WHO) defines it, biosafety is the prevention of unintentional exposure to pathogens and toxins and their accidental release. Therefore, this issue is a matter of extreme importance, while dealing with genetically modified organisms (GMOs). GMOs, as their name indicates are organisms that are not naturally encountered in the environment, and their release to the environment can constitute a safety issue not only to the team members and people that have access to the working space, but it can spread and affect to the institution that hosts the laboratory (in our case University of Copenhagen), becoming a safety problem for the public or for the environment.
   
   Due to the great consequences that might originate from the incorrect manipulation of the GMOs or biological parts, as well as the incorrect use of chemicals or lab equipment it is an obligation for every team member to have enough biosafety knowledge in order to prevent and reduce any potential risk.
   
   This is why; our team followed a lab tour concerning the biosafety in the lab provided by the laboratory technician Lene Daltsen. During this walk our group was informed with the basic rules of the lab as well as with the biosafety regulation at our university. Moreover, we also took part of a workshop concerning Biosafety at the Technical University of Denmark (DTU) hold by professor Chris Workman. Before the workshop every team member had to complete a short safety quiz that allowed us to evaluate our knowledge. At the workshop we develop our knowledge in lab safety procedures and we got familiarized with the database of chemicals ”Kemibrug” that among other information, contains a safety data sheet (SDS) of the chemicals used normally in the lab.
   
   We believe that the knowledge acquired during these two meetings, as well as the knowledge acquired during our university programmes provided us with a good understanding of the importance and correct use of the biosafety procedures in the lab.

2. Biosafety risks concerning

   1. Microorganisms

      Our project uses two different Magnetospirillum species known as M. gryphiswaldense and M. magneticum. These two bacterial species together with the E. coli strain used (E.cloni) have been classified as biosafety level 1 (BL1). BL1 corresponds to agents that are unlikely to cause any disease to humans or animals, and do not result a mayor risk for the environment or the public safety (WHO-Laboratory Biosafety Manual). Therefore we consider that these three biological strains do not represent a huge risk for the team members, public safety or the environment.
      
      The procedures followed while manipulated these organism corresponded to the ones indicated for by the regulation at the University of Copenhagen for a GMO class 1 laboratory. This regulation can be seen bellow.
      
      

      Requirements for work in GMO Class 1 laboratory

      1. Needless traffic in and out of the laboratory must be limited. All visitors must wear a lab coat. Visitor lab coats are located at the entrance to the laboratory.
      2. The laboratory must be kept clean and tidy and in good order. After work, all surfaces must be cleared and decontaminated by the user. In case of GMO spill, immediate decontamination must be carried out.
      3. All biological waste, including non-GMO waste, is discarded in special bins labeled “Biohazard” and “GMO”. The material is sealed before being transported for autoclaving. Containers with liquid waste must be clearly labeled with autoclave tape labeled “GMO-waste”.
      4. Mouth pipetting is absolutely forbidden.
      5. Smoking is absolutely forbidden, as well bringing or storing any kind of food or drink into the laboratory.
      6. Personnel must wash the hands after having been in contact with any type of organisms, GMO or non-GMO, before leaving the laboratory.
      7. Special caution must be taken to minimize aerosol production.
      8. All experiments in the laboratory must be conducted according to this procedure. The procedures are also valid for experiments including recombinant nucleic acids.
      9. Transport of GMO-material between classified areas must be performed using a closed container clearly labeled”Biohazard” and/or ”GMO”.
      10. If the laboratory is no longer to be used for GMO work, down-classification is carried out after main cleaning.

      Other regulations concerning the rules for the correct use of the GMO class 1 laboratory such as the requirements necessary for the use of hume, or cleaning of the laboratory can be seen at the University web page (in Danish).

   2. Biobricks

      MamC gene copies from M. gryphiswaldense and M. magneticum respectively were purchased from DSMZ. They obtained the sequences by molecular cloning, and therefore no additional changes were made to the original sequence. Thus, these gene copies do not represent a mayor risk than BSL1.
      
      Enhanced Green fluorescent protein (EGFP) - the SDS states that this recombinant protein has no hazardous or toxic properties known and in cause with any unprotect contact with it, this surface should be cleaned with water.
      
      Enhaced Flavin Monoucleotide Based Fluorescent Protein (eFBFP) - is a marker for the gene expression that has not been shown to constitute any risk for the human or other animal’s health. Moreover, we think that this gene even if it will transfer to other bacteria from the environment, they will not be naturally selected since it represents an energy waste for the bacterial cells to produce a product that is not useful.

   3. Chemicals

      Ethidium bromide (EtBr), is a potential carcinogenic agent that was used while working with the agarose gels. Therefore extreme care was taking in the gel room (proper use of gloves and dispose of contaminated material), in order to avoid contamination to other areas.
      
      Boric acid (H₃BO₃), is a compound present to the culturing medium of our bacteria, and may affect the fertility in woman. Therefore, it might result in a risk for the team members or personal with access to the lab, so extreme caution and decontamination was adopted while using any surface or object that was in contact with it.

3. Can our new biobricks constitute a safety risk?

   We believe that since the biobricks we used, were not potentially harmful, and since they did not suffer any major changes, they will not represent a safety issue. Moreover, we also used two antibiotic resistance genes (kanamycin and ampicillin resistance genes). In case of unintentional release of our genetically modified bacteria into environment, we think that no event of gene transfer will be possible, since the oxygen is a limiting factor, and their growth is inhibited by oxygen presence.

4. Local Biosafety Group at the University

   The University of Copenhagen treats its responsibility to secure its institution seriously. There are four different Safety Groups that are in charge of all the rooms, laboratories and teaching laboratories at Life Faculty. Moreover twice per year, they have a meeting with the head of the safety department.
   
   The biosafety regulation at the University of Copenhagen can be seen at: http://www.plbio.life.ku.dk/om_instituttet/Intranet/Sikkerhed_arbejdsmiljoe.aspx. These regulations fulfill the Danish legislation concerning biosafety , and are in agreement with the Council Directive of the European Union 98/81/EC on the contained use of genetically modified microorganisms.
   
   The Biosafey committee is familiar with our project and our presence in the laboratory and the only condition they implemented is that we are not allowed to be in the lab without any supervision.

5. Useful advises for the future iGEM competitions

   We think that an introductory workshop or a small reunion about the Biosafety rules at the institution where the projects will take place would be necessary for every team.
   
   Moreover knowing these regulations may not be enough in some research projects depending on their biobricks, microorganism or chemicals, so we also believe in a smart design of the experiments. By a smart design, we consider that while creating your genetically modified organisms, the worse scenarios should be taking into account. Therefore an event tree analysis and fault tree analysis should be carried out. The first type of analysis corresponds on analyzing the consequences of the failure of one part in your system, while the fault tree analysis analyzes the possible failure of the whole system and tries to understand the cause.
   
   After this analysis, solutions should be adopted in order to prevent as many possible failures as possible depending on the experiment. For example one can:

   • introduce suicidal genes
   • delete gene necessary for the production of toxic or potential harmful compounds
   • delete genes required for an amino acid synthesis (that will be supplied in the enriched medium)
   • divide gene clusters, in order to localize the genes on the bacterial chromosome and plasmid, etc.

   These designs will ensure that the microorganisms will not survive on a natural environment or will limit the gene transfer among other bacteria.

6. Potential risk of magnetosomes applications

   Our project can be considered as a small step on elucidating the biogenesis of magnetosomes. Since we propose three different uses for the magnetosomes, we thought it would be relevant to speculate on some of the possible safety risks that they might cause. Therefore, the following section tries to analyze some of the risks concerning the magnetosome use.

   1. Magnetopharma

      The potential use of magnetic nanoparticles to deliver therapeutic agents, and their posterior targeting by using external magnetic field has been already documented (Mikhaylov, et al, 2011). Among the literature, the results assessing the magnetosome and magnetite cytotoxicity seem to depend on the cell type, tissue and magnetosome coating design (Santhosh and Ulrih 2013).
      
      The pathway the magnetosomes appear to follow is that they are engulfed by the cell, where they fuse with an endosome and later with a lysosome, where they are degraded. Therefore their membrane as well as the magnetite present inside are biodegradables. More studies must be performed in order to analyze the changes on the gene expression patterns by the magnetosomes.
      
      Intelligent design of the coating seems to be a requirement for the patient safety, since by designing the coat you can:

      • target directly cancerous cells (e.g. attaching a receptor on the surface)
      • protect the magnetosomes from drug spill caused by temperature or pH changes
      • increase stability and therefore life time
      • get less protein adsorption on the magnetosome surface
      
      We believe that future studies on the magnetosomes biogenesis and coating, will minimize any risk for the patients and that magnetosomes could be used effectively as a drug targeting system, especially since their visualization uses a noninvasive technique such as MRI.
      

   2. Magnetopaint

      This use will not represent a low risk for the public or environmental safety since it will be mainly used in bioart. Therefore we think that since the human contact will be minimal, the main responsibility would be to ensure a correct disposing of the magenetopaint.

   3. Magnetopower

      If magnetosomes could be used for electronic devices such as batteries, an environmental assessment should be performed in order to see if it will present any toxicity to the environment, or if they are biodegradable.