Team:TU Delft-Leiden/Project/Safety

Safety

Overview

The aim of this team TUDelft iGEM 2014 was to build up a novel biosensor capable of emitting an electrical output in response to a signal using the microorganism Escherichia coli. Three modules have been developed in order to facilitate an electrical output and thus electron transport (see the Project Overview ). To make this possible, the following genes have been used:

Table 1: Genes used in this project.

Gene Origin	Gene/ Gene cluster Name	Short Description
Shewanella oneidensis	mtrCAB ccmAH	Transport electrons from the periplasm to the extracellular space Required for the proper maturation and folding of proteins carrying a heme group (such as the MtrCAB proteins)
Escherichia coli	csgAB	Two of the genes involved in the formation of curli
Aequorea victoria	eGFP	Fluorescent protein
Entacmaea quadricolor	mKate2	Fluorescent protein

For both molecular and characterisation work, the organism E.coli has been used. On the following table, the actual strains used have been listed:

Table 2: Bacterial strains used in this project.

Strain name	Classification	Use
DH5α	ML-1	Cloning work
BL21(DE3)	ML-1	Land Mine module characterisation
C43(DE3)	ML-1	Extracellular Electron Transport module characterisation
ΔCsgB LSR12	ML-1	Conductive curli module characterisation

Landmine detection

The use of genetically modified organisms (GMOs) represents some risks and concerns for society. Therefore, their use and handling are strictly regulated. For the development of a Plug-and-Play biosensor based on electrical current, the TU Delft iGEM team has worked on the specific case of landmine detection. Hence, our team has been dealing with chemical components that represent a big risk for human health and for the environment. As a consequence, full awareness of the potential risks of the chemicals used for our experiments was taken, and a detailed protocol was developed to handle safely the aforementioned chemicals.

Hazardous chemicals used

Land mines are mostly based on the explosive compound 2,4,6-trinitrotoluene (2,4,6-TNT). However, these mines many times contain impurities of 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-DNB) which, due to their volatility, can leak out of the mine. Therefore, 2,4-DNT and 1,3-DNB are quite convenient compounds for the construction of a land mine-biosensor. These chemicals, nevertheless, require special attention to ensure a safe working environment.

2,4-DNT

2,4-DNT is classified as Toxic, Harmful and Dangerous for the environment according to EU Directives 67/548/EEC or 1999/45/EC. Of special imporance is its toxicity if swallowed, in contact with skin or if inhaled.

1,3-DNB

1,3-DNB is classified as Very Toxic and Dangerous for the environment according to EU Directives 67/548/EEC or 1999/45/EC. Of special imporance is its fatality if swallowed, in contact with skin or if inhaled. It may also cause damage to organs through prolonged or repeated exposure.

Acetonitrile

For safety reasons, it is better to work with the aforementioned chemicals in liquid solutions. Due to their hydrophobicity, 2,4-DNT and 1,3-DNB were obtained dissolved in acetonitrile. This solvent itself also needs to be handled carefully for it is Highly flammable, Harmful and Irritant according to EU Directives 67/548/EEC or 1999/45/EC.

Safety measures

These chemicals need to be always handled at the Fume Cupboard. A fume cupboard is a piece of laboratory equipment designed to limit exposure to dangerous fumes. The air inside the fume hood is either vented to the outside or else filtered and recirculated.

Additionally, solutions containing either 2,4-DNT, 1,3-DNB or acetonitrile will be handled wearing Butyl-rubber gloves of a minimum thickness of 0.3mm. For more protection, Nitrile-rubber gloves should be worn underneath.

Concerning the disposal of the chemicals, these compounds are toxic to aquatic life and dangerous for the environment. Consequently, they need to be disposed appropriately. After the tests using bacteria together with these hazardous compounds, the bacteria will be first killed by adding an antibacterial agent during the required amount of time. Afterwards, the mixture containing the dead microorganisms together with the land mine compound will be brought to the Chemical Central Waste of the university to be disposed.

And last but not the least, if some chemical is spilled on the gloves, they will be removed as soon as possible without touching the skin with the gloves. In case of emergency (eg. spills) the affected area must be covered with tissues. The room needs to be evacuated for half an hour. When leaving the room make sure you take off and dispose properly the lab coat. When returning in the room, plastic bags for biological waste need to be taken. The waste should be disposed in a chemical waste bin. The Biosafety officer needs to be contacted for reporting the situation.

Safety Proposals

In order to obtain the authorisation from TU Delft Applied Sciences faculty to work with the aforementioned chemicals, an internal safety proposal had to be developed on top of the standard iGEM safety proposal . To read the additional protocol developed by our team click on the following link .

Important Note

The procedure described above was thoroughly reviewed and approved by the chairman of the Safety Committee of the Applied Sciences faculty of Delft University of Technology, Jos van Winsen.