Team:UPF CRG Barcelona/Safety

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SAFETY

Lab work and project safety is a serious issue for our team. In each one of our Bachelor studies, we received lab safety trainings. Apart from this, our instructors gave us safety protocols and occupational hazards protocols, and finally we were introduced to all of the safety equipment of the laboratory space we were provided.

Final Safety Form iGEM 2018

General laboratory safety

When we designed our experimental lines, we decided to work on an open bench with a non-pathogenic strain. We have been working in the safety level of Biosafety level 1.

In the Biosafety level 1 protocol there is included:

  • Personal protective equipment such lab coats, eye protection and gloves
  • Mechanical pipetting
  • No food or drinks in the laboratory
  • No phones or computers in the work bench.
  • Maintenance of the work zone clean. We cleaned before and after using with 70% EtOH.
  • Autoclaving the material used.

In addition, the whole team had previously worked in laboratories of the same biosafety level or higher.

General laboratory safety protocols and precautions

  • Specific safety equipment such as emergency showers and eyewashes, as well as the fire extinguisher or the fire blanket.
  • Waste disposal: Chemicals, biologicals and solids waste disposals.
  • Personal protective equipment. In the lab is needed to wear: lab coats, long pants and enclosed shoes. Do not wear contact lenses.
  • Appropriate storage for both flammable and potentially hazardous chemicals.
  • Appropriate use of potentially hazardous chemicals. We had access to fume hoods.
Cytotoxic residues
Biological residues
Sharp biological residues

Figure 1. The different residues bins that are used in our Research Park. The blue one is for cytotoxic residues, for example the SYBR Safe gels. The yellow ones, the small and the big one, are for biological residues. Everything that has been in contact with a biological part needs to go there. For example the pippette tips after touching LB medium with E. coli

Application safety issues

Our projects aim to be a proof of concept for a possible probiotic. In this way, we have to consider safety and biocontainment in order to guarantee a harmless product. First of all, a non-pathogenic strain was used: Escherichia Coli. The strain being used is a GRAS (Generally Recognize As Safe). We used antibiotic resistance just to select the bacteria who had incorporated the desired genes. However, this approach was only used as a proof of concept for our system. In this way and thinking further in our project as a possible probiotic, we are planning to integrate these modifications inside the bacteria genome. For this reason, we used MAGE (Multiplex Automated Genomic Engineered).

Genetically Modified Organism

In the design of our system we plan to overexpres the own E.coli genes related to the LCFA degradation metabolism. As it has been mentioned, even though antibiotic resistance was used as a way of selection, it was only a step before genome modification.

When assessing our GMO risk the following was considered:

  • Characteristics that potentially could result in harmful effects.
  • Assessment of possible consequence of these harmful effects, if they occur.
  • Assessing the probability of the occurrence of the individual potential harmful effects.

In our case, the unique possible harmful effects derived from our GMO would be the antibiotic resistance genes and their spreading, but as mentioned, this problem will be solved with genome integration. When considering the genes that we were expressing in our device, any of them would constitute a danger for the human body.

Safety expertise

In order to consider the ethic implications of our proof of concept we had a talk with Cristophe Robaglia from Aix-Marseille Université. He aware us about the need to consider possible allergenic when overexpressing membrane receptors. He also aware us of the possible risks and the regulations that our probiotic will need to consider.

Project specific laboratory safety

We have used some chemicals and substances that need special mention of biosafety. We consulted our instructors and checked the Safety Data Sheets provided by the purveyor beforehand.

  • SYBR safe DNA gel stain: We chose SYBR safe because is safer compared to Ethidium bromide. It is less mutagenic, and its sensitivity is excellent. Referring the Safety Data Sheets classifies the substance as not hazardous for health nor for the environment. However, if there is skin or eye contact needs rinsing with plenty of water. Ingestion and inhalation are not expected to present significant presence under normal use.
  • Antibiotics: We used ampicillin, chloramphenicol and kanamycin as the main antibiotics. It is needed to take into account that these chemicals can cause acute toxicity and mutagenicity, so when working with them safety measures were considered.
  • Pyridine: We used pyridine as a pH corrector for the fatty acid detection and quantification assay. Pyridine is flammable, and it has acute toxicity of category 4 in dermal, inhalation oral. Needs to be used in a fume hood.
  • Isooctane (2,2,4-trimethylpentane): We used it as the main component for the fatty acid detection and quantification assay. It is highly flammable, therefore it needs special storage and to be used in a fume hood.
  • Acetone: We used for the fatty acid detection and quantification assay It is flammable and can cause skin dryness or cracking.
  • Cooper II Acetate: We used it as the main component for the fatty acid detection and quantification assay. To work with Cooper II acetate eye protection, mouth-nose mask, gloves and lab coat were needed.