Introduction - Lab Safety

Safety Level

The TU Eindhoven has multiple lab spaces with different Safety Levels. As an iGEM team, access to one of the labs with BioSafety Level 1 was gained,

Within Biosafety Level 1, people are allowed to work with microorganisms which cannot cause any diseases or harm to humans.

for us the ML-I laboratory. In this kind of lab most work involved recombinant expression of protein in E. coli or yeast expression systems, and the application of bacteriophages for phage display.
In the ML-II laboratory, with BioSafety Level 2

Within Biosafety Level disease. It also includes microorganisms which cause a disease for which a vaccine or cure is known. The rules to enter a Biosafety Level 2 laboratory are stricter.

, the work typically involves the use of immortalized cell lines, but also tissues or primary cells from animals or humans. In the ML-II also takes place the genetic modifications of eukaryotic cell lines for addressing localization and interaction of transfected proteins.
During our project a lab with BioSafety Level 1 is sufficient, because only work with the E. coli chassis was preformed, which is a risk group 1 organism.

Safety Training

After a safety training and labtour through the ML-I laboratory the team gained access to the lab. During the training various topics were adressed. For example about the equipment and the biosafety rules in the Netherlands. (which follows the Cartegena- and European protocols.

The most valid Biosafety rules in our lab are:

Keep windows and doors shut.
Keep everything clean and tidy, make sure enough disinfectant is present.
Always wear a marked and closed lab coat, which is not permitted outside the lab. The lab coats have to be sterilized by autoclaving before sending it to the laundry.
Don’t wear any watches or jewellery, or keep them covered by gloves or lab coat. Bags, laptops etc. are not allowed and can be stored outside the lab in lockers.
Avoid any contact between your hands and your face. Don’t eat, drink or smoke inside the laboratory. Storing food or drinks is also not allowed.
Avoid formation of aerosols. Mix and centrifuge in closed tubes. The use of a needle is allowed only when no other method is available.
Pipetting with the mouth is not allowed; use the available equipment (pipette boy or pipette bulb).
Always disinfect your working space before and at the end of your activities.
After a contamination of your working space (e.g. when you spill any biological material) disinfect the working space.
After working with biological agents and when leaving the laboratory, always wash your hands with water and soap.
All re-usable materials that were in contact with biological materials have to be sterilized before being washed or discarded.
Solid biological material is gathered in the red biohazard bags, transferred in blue containers and disposed off as special garbage. Liquid biological waste has to be autoclaved before it can be discarded.
All accidents and spills, and all activities with GMO’s or human material have to be reported in the appropriate logbook.

More information about the Safety rules in our lab can be found here

Lab Responsable

The BioSafety officer, the person responsible for the biological safety at Eindhoven University of Technology, is Moniek de Liefde - Van Beest. The Principal Investigator (PI) is responsible for the biosafety and lab safety of the laboratory/laboratories of his/her department. The PI of our lab is Maarten Merkx. He also has a GMO license, all of our GMO work is done under his license, permit GGO-03-121. For license request about GMO's, the instance to contact in the Netherlands is Bureau GGO.

Getting Started - Lab Equipment Safety

Safety Equipment

When working in the lab it is very important to wear a closed lab coat, long pants/skirts, shoes that cover your toes and safety glasses / goggles. Depending on the materials, no, Latex, Nitrile, Cryro or heat-resistant gloves have been used. The equipment is mostly used to protect the skin and eyes from hazardous material, but also to protect the DNA against DNAses.

Work Equipment

During this project a lot of laboratory equipment has been used such as the use of pipettes and their associated tips, the Bunser Burner, centrifuges, PCR machines, Gel Electrophoresis Apparatus, Spectrophotometer or the incubators. For all lab equipment at least one person is responsible. The list of responsibilities can be found at the entrance of the lab. When a person is going to use the equipment for the first time the list provides you with information who to contact for a safe usage instruction.

Work Area

Our work is mostly preformed at an open bench, but sometimes a chemical fume hood was needed when working with hazardous fumes. To know if this precaution was necessary, the material safety data sheets (MSDS) had to be overlooked.

Waste Disposal

There are three different kinds of waste in our lab, namely normal waste, chemical waste and biological waste (biohazard). The normal waste bin can only be used for waste that has not been in contact with chemicals or biohazard material. The chemical waste is first collected in trashcans which are then emptied in the “wisseldrum”. To dispose this, the form at the website from the BBC has to be filled in for renewing them. The biological waste is being divided into two sorts, solid and liquid. The solids are collected in red biohazard bags and transferred to the leak-free blue hospital containers. The containers are closed, labelled and disinfected, before they are being disposed.

The containers are disinfected with 70% ethanol at the exterior. To dispose them another form has to be filled in at the BBC website.

The liquid biological waste first has to be autoclaved also counts for the glass and instruments which have the risk of contamination. After autoclaving, the glass is placed in the dishwasher for further cleaning.

Chemicals

Before using hazardous chemicals, it is necessary to read the accompanying material safety data sheets (MSDS). For the chemicals needed during this project, it was mostly sufficient to use (Nitril) gloves. In some cases, for example when working with HCl and NaOH to set the pH, it was also necessary to work in the chemical fume hood.

Biosafety

Organisms

During this project different strains of the E. coli organism have been used. For plasmid amplification the plasmids were inserted into Nova Blue (K-12), for protein expression into BL21 (DE3) and for Gibson into NEB 5-alpha. All of these strains can be handled as risk group 1 because they are non-pathogenic laboratory strains as chassis organism.

Vectors

For our project we used the pET-Duet-1 vector, which made it possible to express two membrane proteins while using only one plasmid. Additionally we used the vector pEVOL-pAzF, containing the sequence for an orthogonal tRNA synthethase for the unnatural amino acid, which is necessary for our click reaction.
The final biobricks have been made in the pSB1C3 vector. This vector was also used while working on the InterLab Study, together with pSB1A2.

Antibiotics

For our entire project, we used only two antibiotics, these were ampicillin and chloramphenicol. The resistance for ampicillin is found in the vectors pET-Duet-1 and pSB1A2 and the one for chloramphenicol in the vectors pEVOL-pAzF and pSB1C3. Because we perform a double transformation

During the double transformation both vectors were placed into the same bacteria. These plasmids, each possessing their own antibiotic resistance, make the bacteria resistant against both antibiotics.

, our E. coli bacteria have a resistance against ampicillin as well as for chloramphenicol

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