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From 2012.igem.org

We are following the official iGEM Safety instructions.

We are working at the platform of Institute of Systems and Synthetic Biology (iSSB), which is a research unit of Université d’Evry-Val-d’Essonne. It is fully equipped for bacterial genetics and molecular biology work in terms of devices and has typical Biosafety Level 1 laboratories. Our team is under daily supervision of Prof. Alfonso Jaramillo, the team direction of Bio-Synth Group of iSSB. We receive regular help as well from Prof. Ioana Popescu, engineer and hygiene and security correspondent, assisted by Dr. Dominique Zeliszewski, as well as from Thomas Landrain, a Ph.D. student working on site. All the iSSB research team is always around to help and advise us about good laboratory practices. Safety page of the institute we are working in http://wiki.issb.genopole.fr/index.php/HS:Main

Safety questions

1. Would any of your project ideas raise safety issues in terms of:

Researcher safety

Chassis

We are working with level 1 safety organisms, which are not dangerous for researchers:

• Escherichia coli: is the most commonly used gram-negative bacterial chassis in Molecular Biology. E. coli is commonly found in the lower intestine of warm-blooded organisms (endotherms). E. coli can benefit their hosts by producing Vitamin K2,and by preventing the establishment of other pathogenic bacteria within the intestine. We work with DH5a and TOP 10 that are common laboratory strains, considered as Level 1 Biosafety Containment agent and are non-pathogenic. However researchers are advised to use standard laboratory safety equipment and procedures while handling the cultures including wearing lab coats, gloves, etc.
• Xenopus tropicalis: is a model organism for genetics, developmental biology, cell biology, toxicology, neuroscience and recently for synthetic biology. It is not dangerous for humans.

Dangerous substances All the standard molecular biology protocols we use in our experimentation are not dangerous for the researchers. However, our team works with potentially dangerous substances: Ethidium Bromide (EtBr): is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) for agarose gel electrophoresis. Ethidium bromide may be a mutagen, carcinogen or teratogen although this depends on the organism and the conditions. EtBr intercalates double stranded DNA (i.e. inserts itself between the strands), deforming the DNA. This could affect DNA biological processes, like DNA replication and transcription. Acute health effects: May cause eye and skin irritation. May cause gastrointestinal irritation with nausea, vomiting and diarrhea. May cause respiratory tract irritation. May cause methemoglobinemia, which is characterized by dizziness, drowsiness, headache, shortness of breath, cyanosis (bluish discoloration of skin due to deficient oxygenation of the blood), rapid heart rate and chocolate-brown blood. Chronic health effects: May cause methemoglobinemia, which is characterized by chocolate-brown colored blood, headache, weakness, dizziness, breath shortness, cyanosis (bluish skin due to deficient oxygenation of blood), rapid heart rate, unconsciousness and possible death. May alter genetic material. Hence, we do not wash the gel in EtBr solution. Instead, EtBr is added in minimal concentration to the agar solution before it solidifies. It is strictly observed that gloves are used when handling EtBr or EtBr-containing gel. Gels, EtBr-containing gloves and tips are discarded considering them to be 'Hazardous Waste'.

Auxin extraction and identification methods such as HPLC, LC-MS/MS require using potentially dangerous substances. Lab coats, gloves are mandatory. Laminar hood is used when working with acetonitrile and methanol. Following there is a list of used chemical compounds for the mentioned methods and their potential health effects:

• Acetonitrile: inflammable liquid.
  Acute effects: Hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in case of skin contact (permeator). Severe over-exposure can result in death.
  Chronic effects: The substance is toxic to blood, kidneys, lungs, liver, mucous membranes, gastrointestinal tract, upper respiratory tract, skin, eyes, central nervous system (CNS). The substance may be toxic to the reproductive system. Repeated or prolonged exposure to the substance can produce target organs damage. Repeated exposure to a highly toxic material may produce general deterioration of health by an accumulation in one or many human organs.
• Methanol: flammable liquid and vapor. May be fatal or cause blindness if swallowed. Vapor harmful. Harmful if swallowed, inhaled, or absorbed through the skin. Causes eye, skin, and respiratory tract irritation. May cause central nervous system depression.
  Target Organs: Eyes, nervous system, optic nerve.
• Formic acid: inflammable liquid, causes dangerous burns when get in eye- and skin contact.
  Acute effects: very hazardous in case of skin contact (irritant), of eye contact (irritant, corrosive), of ingestion. Hazardous in case of skin contact (corrosive, permeator). Slightly hazardous in case of inhalation (lung sensitizer). Non-corrosive for lungs. Liquid or spray mist may produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Inhalation of the spray mist may produce severe irritation of respiratory tract, characterized by coughing, choking, or shortness of breath. Inflammation of the eye is characterized by redness, watering, and itching. Skin inflammation is characterized by itching, scaling, reddening, or, occasionally, blistering.
  Chronic effects: Slightly hazardous in case of skin contact (sensitizer). Mutagenic for mammalian somatic cells. Mutagenic for bacteria and/or yeast. The substance may be toxic to kidneys, liver, upper respiratory tract, skin, eyes, central nervous system (CNS). Repeated or prolonged exposure to the substance can produce target organs damage. Repeated or prolonged contact with spray mist may produce chronic eye irritation and severe skin irritation. Repeated or prolonged exposure to spray mist may produce respiratory tract irritation leading to frequent attacks of bronchial infection.
• Perchloric acid: explosive liquid.
  Acute effects: very hazardous in case of skin contact (corrosive, irritant), of eye contact (irritant, corrosive), of ingestion,. Non-corrosive for lungs. Liquid or spray mist may produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Inhalation of the spray mist may produce severe irritation of respiratory tract, characterized by coughing, choking, or shortness of breath. Prolonged exposure may result in skin burns and ulcerations. Over-exposure by inhalation may cause respiratory irritation. Inflammation of the eye is characterized by redness, watering, and itching. Skin inflammation is characterized by itching, scaling, reddening, or, occasionally, blistering.
• Iron (III) chloride: corrosive. Harmful if swallowed. Causes eye and skin irritation and possible burns. May cause respiratory and digestive tract irritation. May cause blood abnormalities. May cause liver and kidney damage.
  Target Organs: Blood, kidneys, liver.
• Sodium hydroxide: Harmful if swallowed. May cause adverse reproductive effects based upon animal studies. May cause liver and kidney damage. Causes burns by all exposure routes.
  Target Organs: Kidneys, liver, cardiovascular system. Causes eye and skin burns in contact, destructive to the tissue of the mucous membranes and airway, chronic exposition can cause lungs and ovaries cancer (tested on hamster).

Public safety

Any part of our project is not potentially dangerous or harmful for public. All DNA constuctions and E. coli expressing resistance genes (e.g. Ampicillin and Kanamycin) are disposed of properly, either by autoclaving or adding bleach.

Environmental safety For our biological waste we are using the special plastic bags which are marked as biohazard, and which are disposed in the specially designated areas. All contaminated waste is treated as 'Bio-Hazardous Waste' and is decontaminated (autoclaved) before being put into a red trash bag or a sealed medical waste box. The bio-hazard bag is placed in a pre-chosen area for picking up. Appropriate measures for waste disposal are taken. Waste is not washed down the sink or thrown into public dust-bins. We are using embryos and tadpoles in laboratory conditions, they will never be released to the environment so we will not cause any environmental safety issues. They are going to be euthanized before reaching their reproductive age.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?

If yes, did you document these issues in the Registry? how did you manage to handle the safety issue? How could other teams learn from your experience?

No, there are no potentially dangerous biobricks in our constructs. All DNA constuctions and E. coli expressing resistance genes are disposed of properly, either by autoclaving or adding bleach.

3. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country? In our host laboratory- iSSB there is no local committee which deals with the biosafety. However there is health and safety officer Prof. Ioana Popescu, Dr. Dominique Zeliszewski and people who are trained in first aid. Emergency numbers and first-aid rules are easily visible in each room of the institute. ISSB is a part of CNRS (French national center for research) .Consequently, the institute conforms to safety rules and regulations set by CNRS. IGEM members have been biosafety-trained proior starting in vitro manipulations. More over, IGEM members have civil insurance in case of any lab accident. The permanent research team members are available throughout the time when experiments are conducted, and they offer supervision every now and then. The laboratory has been classified as to biosafety categories and complies to French law (French Biosafety-National Legislation that deals with biohazards and GMO/GMM). Environmental measures has been considered wastes are being disposed of in a specific containers and thereafter treated in accordance to the type of wastes whether biological or chemical.

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Safety in synthetic biology has always been both researchers’ and public’s concern. As a result, having an IGEM biosafety guidelines set for new IGEMers with what to use versus what is off limits. As for what can be done to make IGEM parts, devices, and systems safer, we propose the following potential solutions:

• Introducing a CRISPR-induced system that targets and degrades plasmids and parts should it escape lab containment. The introduction of short sequences to all parts will trigger the CRISPR system if the antagonist is absent from the culture medium. This way, all parts and devices are restricted to laboratory containment.
• Inserting killing genes (kill switch) as well as antibiotic resistant genes within all the constructs made for bacteria.
• Designing a genetic switch mechanism making the frogs sterile, in case the tadpoles were issued to metamorphose into frogs, which could increase the risk of environmental dissemination.