Overview

In all research, including iGEM projects, the safety procedures and precautions associated with conducting experiments must be taken into account. Nonetheless, additional considerations were made in regard to laboratory safety since we have been working with a non-standard iGEM organism (an organism not on the white list) in our project.

Lab safety

Before starting an experimental project at our university, we had to complete a mandatory safety and risk assessment of experimental work as dictated by Danish law. The assessment aims to create the safest possible working environment not only for us, but also all our colleagues and the facilities we work in and further, to protect the environment. In addition to reading several work safety documents, an extensive quiz has to be passed to be allowed to work in the laboratories of DTU. This also entails identifying risks in the project and how to mitigate them. All this follows the Danish regulation on working with GMOs. A lab safety tour around the premises with two laboratory technicians was also mandatory, and provided us with knowledge of practical procedures and policies for emergencies. Additionally, training from our laboratory technicians was mandatory before using any potentially dangerous piece of equipment in our laboratories.

Our organism of choice

The filamentous fungi Aspergillus niger, sp. ATCC 1015 forms the foundation of our project. This specific strain is classified as Biosafety Level 1, indicating that it is safe to use and handle in a standard microbiology laboratory. A. niger is also used widely for fermentations, and is classified as GRAS (Generally Regarded As Safe) by the United States Food and Drug Administration. As the chosen organism is a spore-forming fungus, it is not on the white list; therefore, a safety check-in has been performed to assess how we would handle it in our laboratory. The spores of A. niger are airborne and can contaminate other samples. Therefore, anytime we worked with the fungus, all our transformations and experiments were performed in a sterilized LAF bench. In addition, the workplaces were cleaned thoroughly with 70% ethanol before and after work to wipe away not only the cross-contaminating substances and organisms, but also the spores.

Furfural

Furfural (Merck Millipore, #804012) is a toxic compound and is produced as a byproduct from the breakdown of xylose during pretreatment of lignocellulosic biomass. It appears as a colorless or pale yellow oily liquid with a penetrating smell, and is soluble in water. Since it may be toxic by ingestion, skin absorption or inhalation, we had to be cautious while handling it. To avoid inhaling vapors and aerosol and getting in contact with furfural we were working with it under a fume hood and wearing gloves and eye protection. We made sure to follow the special instructions given by the official DTU website for identification of chemicals, before we started to use furfural for any experiments.

Lysis buffer

In this project E. coli were lysed by a lysis buffer containing these harmful compounds; sodium azide (Merck Millipore #822335), sodium fluoride (Merck Millipore #106449) and ethylenediaminetetraacetic acid (EDTA) (Sigma #E9884). The sodium azide and sodium fluoride are very toxic if ingested (H300 + H301 + H310 + H330) and to a lesser extent if they have contact to the skin (H373 + H315). EDTA causes eye irritation (H319). This meant that the lysis buffer was handled with caution to minimize the risk for spills. In addition, we wore gloves so as not to get into contact with sodium azide and sodium fluoride.

IMAC purification

Protein purification was performed using immobilized metal affinity chromatography (IMAC). IMAC use imidazole (Merck Millipore #814223), which is harmful when ingested (H302) and causes skin burns and eye damage (H314). It is also very important that not work with imidazole when pregnant, because it may cause damage to an unborn child (H360D). The safe precautions taken were a good presence of mind when working with imidazole and gloves were worn.

Working with gels

Safety precautions when working with agarose gels and stains for purifications were as described in the Experiments section.