RUigem biosafety in the lab

Waksman institute of microbiology Our experimental lab work this summer took place at the Waksman Institute of Microbiology on the UMDNJ campus at Rutgers University. This building is named in honor of Dr. Selman Waksman who was a soil microbiologist and a professor at Rutgers for four decades. In 1952, Dr. Waksman received the Nobel Prize for Physiology or Medicine for his discovery of streptomycin, which is isolated from Streptomyces griseus. This discovery led to his coining of the term "antibiotic.”   Following this discovery and the application of streptomycin as broad range antibiotic, questions regarding the resistance of bacteria in the presence of an antibiotic arose. Consequently, biological safety precautions had to be reevaluated to better reduce hazards and risks in the laboratory.    We believe that adherence to good laboratory practice is beneficial to many, not just the researcher. These qualities can help to avoid errors, allow for experimental procedures to be consistently repeated, and aids in the credibility of the work to others. Throughout the summer, we have carefully worked with Escherichia coli strain DH5a (Genotype: fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17), various antibiotics (ampicillin, tetracycline, kanamycin), chemical reagents and hazardous material. Our safety regulation and laboratory responsibilities included careful labeling schemes, detailed documentation and a cleaning routine of laboratory bench tops before and after work sessions. Waste collection and disposal for specific hazards was also carefully regulated.

safety questions
Our iGEM projects involve the construction of gene regulatory networks that utilize positive and negative feedback loops found in microorganisms. The circuits we have proposed are made up of genetic devices such as transcriptional regulators (activator and repressor proteins) and reporter genes. In the duration of our project we did not come across any DNA BioBricks that could cause pathogenic harm to the researchers, the public or the environment.Rutgers Environmental Health and Safety regulates protocols and waste disposal methods used by federally funded research projects. (REHS, http://rehs.rutgers.edu/lsbio_comm.html)   1. Would any of your project ideas raise safety issues in terms of? Researcher Safety Use of hazardous chemicals: Ethidium Bromide (EtBr) Public Safety None Environmental Safety? None Security Threats? None Our projects are involved with logic circuits and light detection. By themselves they pose no risk to anyone.    2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, For the parts that are already in the registry no safety issues have been encountered.  The two BioBrick parts we are creating from scratch are transcriptional regulators which should not raise any safety issues.   3. Under what biosafety provisions will / do you operate? Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible. Rutgers Environmental Health and Safety (REHS) regulates protocols and waste disposal methods used by federally funded research projects. (REHS:http://rehs.rutgers.edu/lsbio_comm.html) Is there a local biosafety group, committee, or review board at your institution? REHS provides oversight and training for various hazards such as medical waste, hazardous waste, and radioactive waste. Dr Vershon, our PI, reviewed our project for possible safety issues; he did not find any. He also informed us of the labs procedures for disposing waste and general safety procedures.  The procedures have all been reviewed by REHS and are approved under the Recombinant DNA RD-00-029 (11/28/2000 – 12/31/2015) issued to Dr. Vershon by REHS. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible. Yes, the NIH has guidelines for biosafety available here: http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf   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? If extra money is available, Crystal Violet and other dyes are much safer and more accurate alternatives to EtBr. Come up with an alternative to Bleach to clean out E.Coli from the sink and waterways. Use autoclavable/sterile materials instead of gratuitous amounts of plastic every day. Software Approach: Software that scans bricks for potentially hazardous DNA sequences Software that calculates risk of mutation that could create a potentially hazardous DNA sequence. Future plugin for MYSIS that can output a protocol to insert any genetic ‘watermark’ via site-directed mutagenesis

general laboratory rules
guidelines No eating, drinking, smoking, applying cosmetics, etc. in the lab.  Access to the laboratory is restricted to only when Dr. Vershon or Dr. Mead from the Vershon lab is present.  Please do not bring friends into the lab (Dr. Vershon has our roster. If we add new members we will okay it with him before they are allowed in the lab). Work areas should be cleaned after any chemical spill. Mechanical pipeting devices are always used.  Mouth pipeting is not permitted. Hands should be washed before and after working in the laboratory. Disposable gloves should be worn when working with hazardous chemicals such as EtBr.  Lab coats must be worn in the lab to protect your street clothes from contamination.  Lab coats should not be worn outside of the laboratory.  Shorts and sandals are not permitted in the lab. Waste must be disposed of properly (see Waste Disposal on p.1-5). Sterility/aseptic techniques Pipette tip boxes and microfege tube bags must always be tightly closed. Media and reagent bottles must always be capped tightly. Common courtesy If you are using Dr. Vershon’s supplies (i.e. media, plates, reagents) always inform someone when supplies are very low. Don’t take the last bottle! Help out making media when requested. Sterile solutions should not be returned to the general stocks. (Source: iGEM-Lab1-Pipeting; Vershon, 2011)

storage and saving
Incubator (37 C) Plates should be incubated agar side up, properly labeled and documented in the lab notebook! Liquid cultures should be tightly capped, properly labeled and documented in the lab notebook! Turn on shaker after taking off your samples! Turn off light after leaving the room. Room temperature (20 C - 25 C) DNA Kit (with lyophilized DNA) Ethanol Concentrated Buffers Refridgerator (4 C) Plates (agar side up, properly labeled and documented in the lab notebook!) Liquid cultures (tightly capped, properly labeled and documented in the lab notebook!) DNA (i.e. un-lyophilized DNA) Buffers Media Freezer (-20 C) Most restriction enzymes (always read the label!) Low-temperature Freezer (-80 C) Frozen cultures (properly labeled and documented in the lab notebook!)          Light sensitive Tetracycline is light sensitive and should be saved in the refridgerator with foil around it and tape. If a bottle is brown, it is light sensitive and should stay this way. Cleaning up Generally, make sure everything in the lab is in order. Cover things so they may not collect dust. Always close pipet tip boxes and bags with microfuge tubes to maintain their sterility. Keep autoclaved bottles tightly closed. Glassware. All tubes must be washed out in Waksman Room 234. Place the glass tube and the cap in its appropriate basket. Disposables. If it’s made out of plastic and you do not need it, it’s probably garbage. Make sure before throwing out tubes that you are absolutely sure you don’t need it. If you are continuing someone elses experiment and/or you are not sure whether to save the tube, save, label and store it in the refrigerator just in case. Refrigerator/freezers. All refrigerator/freezer contents must be labeled properly and documented in the lab notebook. Tape plates together if they are related. Try not to tape only one side of the stack because if it falls apart or just falls, it gets messy. Reuse tape when you can and again, label the tape. All tubes must be capped tightly. Biohazard (E. coli). Add (a small amount) of chlorine bleach to waste containers and let it sit for a bit before discarding it down the sink.  The drawers are now labeled with respect to their contents, please keep them this way. The window should be shut completely if you have opened it. Place all books back on the shelf. Place all lab notebooks and protocols in the brown binder. Wipe the bench with some ethanol at the end of the day.

References
Barker, Kathy. At The Bench: A Laboratory Navigator. New York: Cold Spring Harbor Laboratory, 1998. Print.   Rastogi, Smita, and Neelam Pathak. Genetic Engineering. New Delhi: Oxford UP, 2009. Print.   Seidman, Lisa A., and Cynthia J. Moore. Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference. San Francisco: Pearson/Benjamin Cummings, 2009. Print.   Nelson, David L., Albert L. Lehninger, and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W.H. Freeman, 2008. Print.   Vershon, Andrew, Barth Grant, Bryce Nickels, and Janet Mead. Introduction to Research in Genetics Laboratory Manual, Spring 2011. Print.