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From 2011.igem.org

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Laboratory Responsibility

The Biosafety and and BioSecurity of all campus labs including the iGem team lab is under the oversight of Towson University's Institutional Biosafety Committee. See this list: Towson University IBC.

These individuals on the IBC play a key role in making sure that all labs at Towson University implement the Institutional and Federal regulations regarding Biosafety and Occuational Safety.

In addition, lab accountability is expected of all individuals working in Towson University Labs, and covers:

• Labeling and documentation - on lab doors, cabinets/storage, waste containers and materials
• Material Data Safety Sheets on any incoming or outgoing lab material.* Project Safety Analysis - where hazards are identified in every lab process and risk reduction strategy is implemented.
• See also the links under the Hazardous Chemicals Sub-section of theTeam:Baltimore/Solutions Page.
• Housekeeping of the facilities and equipment
• Emergency response also falls under the responsibility of the lab from First Aid, Protective devices, training and evacuation routes
• See also the Team:Baltimore/Working under the Hood page.
• Also very important is the lab risk analysis - evaluating the biosafety level (BSL)designation.

The iGem lab is equipped for a BSL 2 designation. While the E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans, we will also use a B- strain for expression of the Taq polymerase when we are assessing the activity or our BB part. This is a BSL 2 designated organism, and we will be taking all necessary precautions when working with the BL21 (D3) strain of E. coli. These include:

1. Wearing complete protective clothing - eye protection, face masks, aprons or lab coats, gloves, covered legs and closed-toed shoes.
2. Taking appropriate care when disposing of the expendibles that are used while working with this organism - like pipet tips, kim wipes, gloves, tubes, etc. These will all go into the Biohazardous waste containers. This waste is autoclaved before disposal.
3. Training all members of the iGEM Team to safely work with this organism. Each student must undergo several hours of afety training, and signs a document containing a checklist of the information covered. See the TU Lab Safety Checklist.

Note: For any safety issues regarding the DMSO, For more information please click http://www.dmso.org/articles/information/muir.htm

Individual Team Member Responsibility

Each team member is responsible for their own safety while working in the lab as well as for those around them. It is required that all team members abide by government, institutional and lab regulations and policy including personnel safety practices and lab safety practices as emphasized in training. Baltimore iGem team members are committed to staying up to date on current regulation and reducing risk for Physical, Chemical and Biological hazards

iGEM TEAM SAFETY CODE

We recognize the importance of each member's personal responsibility to the safety and security of our labs and our work. This includes the duty of not ignoring another's unsafe or possible harmful actions. We are committed to acting in a responsible manner and taking a pro-active approach to staying current with international and national laws, regulations and guidelines. We are dedicated to being informed about principles and practices designed to prevent hostile use of our labs, equipment, materials and products. It is our duty to contemplate the safety and security issues that may arise as an outcome of our projects.

1. • The Baltimore iGEM team is a diverse group of students with a wide range of ages and varying backgrounds – all with a common interest of genetic engineering. The main Baltimore iGEM team lab is located at the Towson University under the direction of Dr. Elizabeth Goode. The responsibility of Bio-Safety and Bio-Security extends from the institution to the lab and to each individual team member. The college is accountable for: hazard identification, a written Hazard Communication Plan (HCP), management of Material Safety Data Safety Sheets (MSDS) and safety training.
   • Towson University abides by federal regulations and guidelines developed and enforced by: Center for Disease Control (CDC), National Institute for Health (NIH), Occupational Safety and Health Administration (OSHA), EnvironmentalProtection Services (EPA), Department of Transportation (DOT) and the Nuclear Regulatory Commission (NRC).The purpose is to provide all those utilizing college lab facilities with a reference guide to working with hazardous chemicals. This program includes a chemical hygiene plan (CHP) detailing chemical safety information and procedures.

   Items included in the CHP are

   General chemical safety rules and procedures

   Purchases, distribution and storage of chemicals

   Environmental monitoring

   Availability of medical programs

   Maintenance, housekeeping and inspection procedures

   Availability of protective devices and clothing

   Record keeping policies

   Training and employee/student information programs

   Chemical labeling requirement

   Accident and spill policies

   Waste disposal programs

   Emergency response plans

   Designation of a safety officer

   • Each iGEM team member is required to complete lab safety training and signs a safety procedure agreemen. Included in the training are Standard or Good Microbiological Practices (GMP) which are basic practices for working with any microorganism. Also some universal lab safety rules are required practice.

Also, we have general lab safety guidelines that include the following: No eating or drinking in the lab; no taking medicine in the lab No applying make-up or lip glass/stick in the lab Closed toed shoes and lab coats/aprons at all times Gloves at all times when handling chemicals and biological and performing experiments/ procedures and mixing solutions Goggles and face masks as needed Fire extinguisher is in the lab, as is an eyewash A full-body safety shower is on Floor 2 of the Smith Hall Science Bldg on the Towson University campus

NEW SAFETY AND SECURITY QUESTIONS

We address Question 3 first: Is there a local biosafety group, committee, or review board at your institution? YES

See the Towson University IBC Member list - the Towson University Biosafety Committee.

Now we go back to discuss the hazards posed by the products or reagrents/ biologicals that we use in the course of our experiments for our iGEM Project:

Would the materials used in your project and/or your final product pose hazards? We do not see our final product as posing a hazard – because the final product for us in the “wetware” side of genetic engineering is a plasmid containing the gene for Taq polymerase – an enzyme currently one of the most commonly used enzymes in the field of synthetic biology. But, there are dangerous substances that we use in the lab in order to produce the desired product.

We use glacial acetic acid for protein purification. We use Ethidium bromide for DNA visualization after gel electrophoresis, and we will use a strain of E. coli that is an ingestion hazard – specifically, the BL21 (D3) Protein Expression E. coli from Agilent.

We start with the BL21 E. coli: The iGem lab is equipped for a BSL 2 designation. While the E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans, we will also use a B- strain for expression of the Taq polymerase when we are assessing the activity or our BB part. This is a BSL 2 designated organism, and we will be taking all necessary precautions when working with the BL21 (D3) strain of E. coli. These include:

1. Wearing complete protective clothing - eye protection, face masks, aprons or lab coats, gloves, covered legs and closed-toed shoes.
2. Taking appropriate care when disposing of the expendibles that are used while working with this organism - like pipet tips, kim wipes, gloves, tubes, etc. These will all go into the Biohazardous waste containers. This waste is autoclaved before disposal.
3. Training all members of the iGEM Team to safely work with this organism. Each student must undergo several hours of afety training, and signs a document containing a checklist of the information covered. See the TU Lab Safety Checklist.

Now we will first address question 1 (a)-(d) and 2(a) forglacial acetic acid and Ethidium Bromide.

Glacial acetic acid:

Corrosive, flammable, poses health risks at these levels: Flammability 2 (combustible liquid flash point 100-200 degrees F), Health Risk 2 (may be harmful if inhaled or absorbed), Contact Level 3 (Severe – protective equipment, and Reactivity is Level 2 (unstable or may react violently if mixed with water), according to the manufacturer of the glacial acetic acid used in our lab (Baker, Phillipsburg, NJ).

(1)a. Risks to safety and health of team members or others in the lab:

This material is used under the hood in our lab, with the sash down as far as is feasible, to reduce the possibility that the handler of the acid will suffer inhalation injuries, which can produce “severe irritation of the respiratory tract, characterized by coughing, choking or shortness of breath,” or splash or spill injuries, which can be severe.

Goggles are used when handling this acid or any corrosive, even if the reagent is also under the hood, behind the lowered sash. If bacteria or other living hosts are exposed to this substance, we really don’t know what might happen. Because of potential teratogenic effects, we would not have a pregnant Team member handling glacial acetic acid. In fact, that will be the case for any of the hazardous substances in our lab.

b. Risks to the safety and health of the general public if released by design or accident:

Accidental release measures include these: If this agent gets in one’s eyes it can, according to the MSDS for the substance, cause “inflammation of eyes, characterized by redness, watering and itching.” We would wash with water for at least 15 minutes. We do have an eye wash faucet installed in the lab.

Skin contact is very hazardous, as indicated by the Contact Safety Level 3. Rubber and latex gloves are insufficient when handling this because glacial acetic acid penetrates rubber gloves. Therefore we use nitrile gloves when working with this chemical. The “skin inflammation is characterized by itching, scaling, reddening, or occasionally blistering,” according to the MSDS for this substance.

If accidently ingested, we should not induce vomiting. We would call 911. Water can be administered as long as the victim is conscious.

There are potential chronic health effects. Information about any carcinogenic effects is unavailable for this substance. There is a risk of mutagenic effects for mammalian somatic cells and bacterial or yeast cells. This means we are careful not to expose any our hosts to this material.

In the case of a small spill: “Dilute with water and mop up, or absorb with some inert dry material” like paper towels or sawdust and place in a hazardous waste disposal container.

On a larger scale, one must heed the fact that glacial acetic acid is flammable and poses a fire/explosion risk. This substance has a flash point at 37.8o C or above. Basically, this means the chemical should be stored in conditions that will never include the possibility of getting that warm. In particular, in this lab, we keep it either under the hood while working with it, or in the fridge or freezer for storage. We would never leave this out in the open in a room containing heat-emitting equipment that might lose A.C., i.e. in a place where the temperature could get up to the flash point. We would never use this on the bench next to a Bunsen burner (or anything for that matter, because we would never use this on the bench – only under the vapor hood.)

c. Risks to environmental quality if released by design or accident:

Toxicological information from the MSDS says ecotoxicity (i.e. it is toxic) in water (LC50) at these levels : 423 mg per liter per 24 hours for goldfish, at 88 parts per million per 96 hrs for Fathead minnows, at 75 ppm over 96 hrs for Bluefish, and at a level >100 ppm over 96 hours for Daphnia. The upshot is we don’t want glacial acetic acid going down our drains, especially because we are not only in the Chesapeake Bay watershed, we are very close to the Chesapeake Bay.

d. Risks to security through malicious misuse by individuals, groups or states:

If this substance is released into a water supply, it could be toxic even if present at low levels as indicated by the research done on fish. If released in vapor form into a ventilation system it would cause acute respiratory distress, damage to eyes and the skin of anyone exposed to that air supply. Theoretically, it could be used as a poison that could have extremely harmful effects - for example, if added to a punch bowl at a party. Even if not swallowed after it was put one’s mouth, the damage to the mucous membranes, nasal passages, and gums would be extreme. If actually swallowed (ingested), the effects are potentially extreme. This substance is known to be a mutagen, corrosive to be toxic to fish at levels around 100 ppm over 24-96 hours.

2 a. The answer to 2(a) is embedded in the discussions presented in the answers to questions 1(a)-(d) for Glacial Acetic Acid.

Ethidium Bromide

Health rating = 3; Fire = 0; Reactivity = 0 Ingestion hazard. “Toxic by inhalation – can cause irreversible effects” according to the MSDS for Ethidium Bromide solution that Promega provides (2011). (See the MSDS from Promega.) Promega also warns that Ethidium bromide solution is hazardous in case of ingestion. The warnings from Promega in its MSDS indicate that Ethidium bromide poses a danger that is significantly more severe than is indicated by the MSDS offered by Sciencelab.com, Inc. in Houston, for 100% Ethidium bromide, which we understand to be in powdered form. According to Sciencelab.com, Inc., this substance is Hazardous in case of ingestion, but only “slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation.” We found it strange that Sciencelab’s MSDS says also that carcinogenic, mutagenic, teratogenic and developmental toxicity are all “not available,” but also states that it “may alter genetic material.” (See the MSDS from Sciencelab.com.) The Health rating from NFPA is 3, according to both Sciencelab and Promega. We noted that the MSDS from Sciencelab was published several years before that offered by Promega, and we heeded Promega’s more severe warnings because of that and because of the general agreement among all of the biochemists and molecular biologists that Dr Liz has worked with that indeed, Ethidium bromide poses serious health risks in case of skin exposure, inhalation and of course, ingestion. The Fact Sheet published by Indiana University states that EtBr is a “potent mutagen and moderately toxic after acute exposure,” lending even more support to Promega’s MSDS warnings. We did find it curious that according to Promega, EtBR is “colorless.” We assume this is some kind of error, as it is obviously reddish-brown in color.

(1)

a. Risks to safety and health of team members or others in the lab:

      Because of hazard through skin contact, when handling the solution we use gloves, coats, closed-toed shoes, safety googles – all the standard gear required to eliminate splash risk to skin and eyes. We use a closed UV box that automatically switches the UV off if opened  and we are very careful after viewing a gel to wipe the gel camera screen and avoid contaminating the keyboard and doorknobs, light switches, etc. in the camera room. The gloves and paper towels are disposed of in the hazardous waste disposal container. We devote one covered container for staining, one for transporting the gel, and one spatula, and when rocking a gel in the E.B. solution we make sure the staining container is covered, labeled and dated. These 3 items (staining container, transporting container and spatula) are used for nothing else in the lab, and are clearly labeled and stored on paper towels on a shelf right next to the rocker. 
          

We make it a practice to never walk around the lab with the container of solution. If we need to refresh the Ethidium bromide solution we bring the bottle of solution to the container rather than moving the container to another location, like a bench. We use painstaking care to prevent any part of the pipetman from touching any part of the bottle, or its lip, when extracting the fresh Ethidium. We store the concentrated Ethidium bromide solution in a well-labeled container in the refrigerator, which satisfies the guidelines that it be stored in a cool dry place away from oxidizing agents. (See the Indiana Fact Sheet for EtBr.)

When mixing the solution from Ethidium in powdered form, we are very careful as well. The scoop is carefully rinsed with a water bottle’s squirt stream, and collected into the weigh cup that was used to weigh the powder. This is disposed of in the hazardous waste bin, as are the kim wipes and paper towels used to dry the scoop. We are careful to wipe down the scale. A paper mask is used to cover moth and nose while working with the Ethidium in powdered form.

Since Ethidium bromide is light-sensitive, exposure to light for several weeks is known to make it inactive for the purpose of intercalating between the base pairs of dsDNA, however, we have found no information regarding the toxicity of the solution after it no longer has the intercalation capability. Because of this, we dispose of used solution only after it has become too dirty to continue to “spike” with fresh Ethidium, and it goes into the hazardous waste container. We have found that there are always ample paper towels and other absorbant material in the hazardous waste bin that we can simply pour the small amount of liquid left after evaporation onto those absorbant materials. We investigated the possibility of putting charcoal into the used solution to allow the charcoal to absorb the Ethidium solution, for disposal then into the hazardous waste bin, but this is not a practice that is done in the labs at Towson University.

b. Risks to the safety and health of the general public if released by design or accident:

Ethidium Bromide, if “released” by design or accident, would be a dangerous hazard to any person who experienced acute exposure. All of the risks – mutagenic effects, toxicity, etc., as described in part (a), would apply to any members of the general public who were exposed in the case of an accidental or any other kind of release.

The bottle of Ethidium powder is stored in a locked cabinet, because if the bottle were carelessly opened by a student or other individual, it could result in contamination of the floor, bench, and surrounding area. This kind of spill would require immediate and thorough attention, and we simply avoid that possibility by keeping it locked away.

c. Risks to environmental quality if released by design or accident:

We would NOT want this material to go down a sink drain or enter the regular sewage system. Because Ethidium Bromide is considered highly toxic, as indicated by the Health rating of 3, if released into the regular water sewage system in Baltimore (Towson is just a northern extension of the city of Baltimore), this substance would enter the Chesapeake, posing a risk to the shellfish (crabs) that make Baltimore famous. There would be no practical way to recover the chemical from the watershed. The only fortunate thing, if “fortunate” is a reasonable word to use in this context, would be the fact that Ethidium is light sensitive, and would change in chemical nature after exposure to the open environment.

d. Risks to security through malicious misuse by individuals, groups or states:

Everything stated above applies here, but it seems unlikely that EtBr would be a “poison of choice,” so to speak, for any group wishing to cause harm to others. Not to say that it would be impossible to use it as a toxic substance with the intent to do lasting harm to others, but there are definitely other substances that would pose more serious long-term health risks – like any agents that are easily dispersed either through water or air, or via contagion.

2 a. The answer to 2(a) is embedded in the discussions presented in the answers to questions 1(a)-(d) for Ethidium Bromide.

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?

Answer: A common training program, such as CDC Lab BioSafety certification, would be useful training to iGEM team members, especially those teams that are laking institutional support. .