Team:Tec-Chihuahua/Safety

] Erwinions

Personal Protective Equipment

When being inside the lab, we have to follow certain rules, these to diminish the dangers that working inside a lab might represent. The rules go as follows:

We always wear lab coats, nitrile gloves and masks avoiding contact with dropplets, aerosols and other contaminants resulting from experiments.

Our personal belongings stay away from the lab when handling infectious materials.

The PPE should never leave the lab, unless it's appropriately packed.

We should always disinfect our hands with 70% ethanol, applying it before and after we've put on the gloves, as well as washing our hands when entering the lab and after leaving it.

Biological Safety Cabinets

We have 3 BSC, they protect us from the microorganisms we work with, as well as protecting the samples from the contamination we or other external agents might cause. As a common protocol, every single time we are about to use one of these cabinets, we should turn on the UV light for 15 minutes minimum, clean our hands and the materials we will introduce with 70% ethanol. As a final step, when done using it, we should clean it and let the UV light sterilize it for another 15 minutes.

Sterile halo: Using burners to create sterile zones

We count with two types of burners, nevertheless, they are used when working in an open bench. Having a burner in the working area is excellent to create a sterile zone, this to prevent cross-contamination among the samples we are working with.

Meker-Fisher burner

Bunsen burner

Lab Access and Waste Management

The labs we work in are categorized as Containment Level 2 Laboratories. They count with lockable doors. These labs are for Biotechnology alumni and teachers exclusive use.

Usually we have to dispose of four types of wastes. They should be appropriately disposed in order to avoid every possible accident a confusion might cause. Sharp objects go in a thick red plastic bottle, biological wastes, such as fluids and organic tissues go in a yellow hermetic bottle. The solid wastes should be disposed in a yellow bag and all of the other materials that were in contact with bacteria or infectious agents, in general, go in a red bag.

Red bottle for sharp objects

Yellow bottle for non-solid biological wastes

Yellow bag for solid biological wastes

Red bag for infected materials

Paenibacillus larvae & Melissococcus plutonius

Trying to develop an effective treatment for the American and European Foulbrood diseases has brought us to work with its causative agents: Paenibacillus larvae and Melissococcus plutonius. Getting to know the main characteristics of these bacteria and developing the right containment methods to avoid accidental release has become indispensable for the correct development of our project.

Paenibacillus larvae is an endospore-forming, facultative anaerobe, gram-positive bacteria; Melissococcus plutonius is described as an obligate anaerobe, gram-positive bacteria. Both microorganisms are characterized for having an important host specificity, being honey bee larvae their principal target organism. Although this turns them into non-human pathogens, they are still the cause of an important infection in honey bee colonies and, consequently, the standard bacteriological procedures were considered during its manipulation in the laboratory.

Biological safety and biocontainment practices included wearing laboratory coat, gloves and eye protection, regular cleaning of work clothes, washing our hands after working with the pathogens and before leaving the laboratory, decontaminating work surfaces after completion of work and after any spill of the bacteria, leaving our personal belongings and food away from the lab when handling infectious materials and regular decontamination of laboratory equipment and materials. It should be noted that handling of these bacteria was done by lighting a burner in the working area or in an ESCO Class II biosafety cabinet. This cabinet was only used after its cleaning with ethanol 70% (v/v) and exposition to UV light for fifteen minutes. The same procedure was always done after the manipulation of the pathogens.

It is important to mention that the strains of these bacteria were supplied by the Research Center in Food and Development (CIAD), located in Cuauhtémoc, Chihuahua. At this center, the correct handling of these pathogens has already been standardized and all the containment methods and protocols were provided to the team.

Choosing a non-pathogenic chassis

The application of appropriate biological safety and biocontainment principles and practices are indispensable for our team. This is the main reason for choosing a non-pathogenic chassis. The chassis we selected to use were DH5a and Top10 for plasmid propagation and BL21 DE3 for protein expression. The three of them can be handled at Biological Safety Level One (BSL-1) and they're also not known to consistently cause disease in immunocompetent adult humans and present minimal potential hazard to laboratory personnel and the environment.

In spite of the low level of risk associated with the biological material, the work with the biological material is performed inside a biological safety cabinet (BSC) whose surfaces are cleaned and disinfected before and after handling the bacterias.

Choosing parts that will not harm humans / animals / plants

Focusing on the production of antimicrobial peptides, our project required the use of the genes that encode these proteins of our interest, specifically, defensin 1, defensin 2, apidaecin and abaecin. These genes come from two different sources; abaecin and defensin 1 are already part of the iGEM Parts Registry, whereas defensin 2 and apidaecin sequences were taken from the Apis mellifera genome reported on the National Center for Biotechnology Information (NM_001011638.1). Later on, these sequences were completely synthesized using the IDT sponsorship. In sum, all of our genes encode antimicrobial peptides that are part of the Innate Immune System of Apis Mellifera. These peptides have bactericidal activity, however, they don’t represent a risk for humans, animals or plants.

Safety Courses

Before starting any of the lab work, we completed courses provided by the Public Health Agency of Canada web page. In these courses, the whole team learned about the proper way to work in a Physical Containment Laboratory Level 2. All of the courses certified below were comprehended by the team to avoid any mistakes made inside the lab, preventing at the same time, accidents that could endanger someone.