Week 1: Principles and Practices

Summary

The following homework was compiled collectively from individual work.
Authors: Thras(me), Erik, Ned, Esther, Melee, Viirj, Jifei, Lining.

Priciples and Practices Case Study:
Expressing GFP in a cell-free system in a community bio-space at Cambridge, MA

1. BL1 lab status requirements (national and international)

BL1 is a set of procedures/requirements for a lab space that are suitable for working with low-risk biological agents. Generally, E. coli K12 strains (e.g. most bacteria used for cloning of DNA and simple synbio experiments) can be used in a BL1 lab, without requiring BL2 procedure to be implemented.

Cambridge defers to the NIH Guidelines and the CDC’s BMBL (they use the BSLn terminology) for how a bio lab should be set up in the Cambridge rDNA ordinance. The Cambridge Biosafety Committee also regulates any biological work done in Cambridge. Their policies and procedures are outlined in more detail than the rDNA ordinance. Esther has more details on the Cambridge situation below.

It might be beneficial to pre-emptively be in contact with people who might be interested in our work, e.g. the FBI. iGEM has representatives from the FBI present at every Jamboree.

The NIH Guidelines “...specify the practices for constructing [but not synthesizing] and handling:

  1. recombinant [and synthetic] nucleic acid molecules,
  2. synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and
  3. cells, organisms, and viruses containing such molecules.”

There are many specific exemptions and restrictions listed in appendices.

What is considered a recombinant (or synthetic) nucleic acid molecule? They are

  1. molecules that a) are constructed by joining nucleic acid molecules, and b) can replicate in a living cell (i.e. recombinant nucleic acids)
  2. nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules (i.e. synthetic nucleic acids),
  3. molecules that result from the replication of those described in (i) or (ii) above.

Note that the highlighted “and” is crucial -- non-replicative DNA is fine. However, DNA is not considered to be replicable if the DNA “does not contain an origin of replication or contain elements known to interact with either DNA or RNA polymerase.” This means that any DNA used for in vitro transcription falls under NIH guidelines, since it must contain promoters that interact with RNA polymerase. In vitro translation (of mRNA) is a different case, since there are no elements that interact with DNA or RNA polymerase. So theoretically you could buy mRNA (or make it somewhere else that falls under NIH guidelines) and do whatever you want with it without being under the control of NIH guidelines.

Base-pairing “with naturally occurring nucleic acid molecules” is unclear -- is sequence-specific base-pairing meant or just any possibility to form any length of base-pairing? The NIH guidelines define four Risk Groups for organisms and viruses. These are the basis for the four physical containment levels, BL1-BL4. Risk Group 1 agents “are not associated with disease in healthy adult humans” and they and their DNA can be handled in a BL1 laboratory. The guidelines also outline biological containment option to limit the spread of organisms or their nucleic acids outside the laboratory.

When there are multiple sources of nucleic acids, the individual sources must be evaluated for their risk group, taking into account that there might be synergistic effects. Throughout the document, the guidelines use the amount of genetic material used as the basis for any decision making (e.g. ⅔ of a an eukaryotic viral genome can be used in a BL1 containment level), as well as “predicted” or “designed” functions, both of which leave a lot to be desired, but is probably practical.

BL1 containment is appropriate for dealing with components derived from non-pathogenic organisms. The scope is also expanded to working with plants and mice, with some additional rules. Culture amounts must be below 10L and certain toxins cannot be encoded in the genetic material (based on lethal dose properties). The BL1 containment level requires a minimal set of procedures to be followed, including daily decontamination of surfaces, regular hand washing, not allowing food to be consumed in the lab space etc. BMBL defines biosafety levels BSL1-BSL4, similar to BL1-BL4. The WHO has a similar set of four risk categories as NIH.

2. BL2 lab status requirements (national and international)

Place

  • Lockable doors
  • Impermeable work surfaces
  • Sealed/locked windows
  • Access to an autoclave
  • Hand washing station (sink, soap/alcohol gel and paper towels)
  • Eyewash
  • Load bearing furniture (not covered in absorbent material)
  • Negative air pressure (can be confirmed by the Biosafety Officer)

Equipment

  • BSL1 + autoclave, directional airflow, no recirculation, disinfection/decontamination procedures in place.
  • Autoclave tray.
  • Bench top sink available for hand washing.
  • Biohazard waste container/bags.
  • Dust pan and broom.
  • Sharps container.
  • Secondary container for transporting biohazards (sealed, leak proof and labeled as biohazard).
  • Certified biosafety cabinet.
  • Vacuum trap for liquid waste.
  • Class I or II Biological Safety Cabinet (BSC) or equivalent containment for manipulations with potential for aerosolization or splashing
  • HEPA filtered vacuum line.
  • Method for decontaminating pipets
  • Recommended: Designated cloth or disposable lab coats for viral culture
  • Recommended: Centrifuges with sealed rotors or safety cups to contain aerosols

PEP

  • Disposable gloves
  • Lab coats
  • Face and respiratory protection may be needed based on procedures and agents

Supplies

  • Disinfectants (e.g. bleach and ethanol)
  • BSL-2 placards (Provided by Biosafety Officer)

Procedures

  • All liquid waste should be inactivated prior to disposal. Bleach should be added to a final concentration of 10% and allowed 30 minutes to inactivate infectious agents.
  • Solid waste should be decontaminated with 10% bleach and/or autoclaved prior to disposal.
  • Standard microbiological practices/aseptic technique (BSL1).
  • Limited access, ‘Biohazard’ signs, ‘sharps’ precautions, defined procedures for Regulated Medical Waste (RMW) disposal and medical surveillance (as needed).

Dangers

  • Agents: associated with human diseases of varying severity, e.g., Hepatitis B and C, HIV, S. typhi, human retroviruses, S. aureus. Includes recombinant DNA activities using viral vector systems such as Adenoviruses and some Retroviral vectors, particularly Lentiviral vectors, and expression of recombinant DNA in BSL-2 organisms.
  • Transmission: inoculation and other percutaneous injuries, ingestion, mucous membrane exposure
  • Various bacteria and viruses that cause only mild disease to humans, or are difficult to
  • Contract via aerosol in a lab setting

Overall differences

  1. Laboratory personnel have specific training in handling pathogenic agents and are directed by scientists with advanced training.
  2. Access to the laboratory is limited when work is being conducted.
  3. extreme precautions are taken with contaminated sharp items
  4. certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

3. How to get Cambridge City Hall approval

The Cambridge Biosafety Committee (CBC) has an “RDNA Ordinance” document that says:

  • Recombinant DNA (RDNA) is: any outside DNA that is put inside a living organism that is then allowed to replicate that DNA.
  • All persons proposing to use RDNA must obtain a permit from the Commissioner with the approval of the CBC.

Source

Application Requirements:

  1. Form an Institutional Biosafety Committee (IBC) -- must include at least one community representative (without personal or business relations with the space), approved by the Health Policy Board.
  2. A health and safety manual containing:
    • all procedures relevant to use of RDNA at all BSLs.
    • a program for waste disposal in compli)ance with fed/state/local laws.
  3. Training program of safeguards and procedures for people using RDNA.
  4. Medical surveillance program determined by the IBC (to be approved by CBC)- e.g. relevant vaccines, occupational health advice, exposure treatment.
  5. Rodent and insect control program.

Source

Actions we should take:

  1. Submit application form and other required documents electronically.
    Contact: Sam Lipson, slipson@challiance.org or 617-665-3838
  2. Give a scheduled presentation of the application materials.
  3. Arrange a site visit to the facility by the CBC.

Annual Actions:

  1. Pay annual permit fee
    $325/yr for 0-10,000 ft space, $650/yr for 10,000-40,000 ft, $1300 for >40,000 ft.
  2. Submit annual report of IBC meeting minutes: protocol summaries reviewed by IBC since last report; IBC membership with emails, home phones, home addresses; major changes to biosafety manual / floor plans.

Source

4. Biohazardous waste disposal

In terms of Biohazardous waste disposal,

Locally: The Cambridge Biosafety Committee through the Cambridge Public Health Department requires that anyone submitting application for permits for laboratory space in Cambridge have access to copies of and have read the National Institutes of Health guidelines and the Center for Disease Control’s Biosafety in Microbiological and Biomedical Laboratories as well as a comprehensive laboratory manual for use in our lab that addresses waste disposal procedures. For our purposes we can use the World Health Organization laboratory manual provided on the class website documentation for application that includes a floor plan of the laboratory space with a designated section for “waste storage floor area” in square feet A description of the protocol and procedure implemented in the lab space in accordance with 105 CMR 480 - Storage and Disposal of Infectious Biological or Medical Waste or the The Massachusetts Department of Public Health’s Minimum Requirements for the Management of Medical or Biological Waste (State Sanitary CODE Chapter VIII)

From 105 CMR 480.100: (G) “All medical or biological waste, except from home sharps users, must be treated on-site or transported off-site for treatment at a minimum once per calendar year” This requires disposal of: blood and blood products (not blood saturated materials) which may be disposed of directly into our industrial sink unless CBC says otherwise and can otherwise be made non infectious and disposed of in sanitary landfills Blood Saturated Materials, Cultures and Stocks of Infectious Agents and their Associated Biologicals, Dialysis Waste and Laboratory Waste which can be rendered non infectious and disposed of in sanitary landfills or properly stored in a code compliant container stored per code and disposed of off site Sharps which can be disposed of by incineration or rendered non hazardous and disposed of in a sanitary landfill. For our space and purposes should be collected in a safe and properly labelled container and disposed of off-site

Biotechnology By-product Effluents: BSL1 or BSL2 facilities may allow biotechnology by-product effluents that contain RG1 or RG2 agents to be removed from the site prior to treatment if the facility meets plumbing codes and meets a set of requirements. These requirements start with establishing an Institutional Biosafety Committee which we may want to figure out how to implement.

Nationally: National Institute of Health Guidelines: Multihazardous Waste ex. “Methanol/acetic acid solutions from electrophoresis procedures containing radioactive material” Avoid generating, minimize volume of the waste generated, call appropriate source for waste management info prior to beginning work. To dispose of waste 1st implement inactivation of wastes such as through autoclaving and properly secure, label, and tag in containers placed in drip trays for removal. Medical Pathological Waste (MPW) If possible convert items to general waste through decontamination (with appropriate cleaning products) or inactivation. Can be contained in boxes and kits acquired through NIH for disposal. Labware should go through chemical decontamination for 30 minutes in an appropriate disinfectant solution and the disinfectant solution should be collected as chemical waste
Internationally World Health Organization: To facilitate proper waste disposal the WHO manual asks those participating in the lab to ask “The principal questions” addressing whether the materials to be disposed have been decontaminated by an appropriate means and whether they have been contained and packaged properly for transfer and to insure no additional harm will come during the process of transfer to an outside source To handle waste we would decontaminate by autoclave if possible for the space and dispose of waste in receptacles labelled properly with categories that adhere to international standards and definitions and the containers must meet to standards to properly house the items being container (ex. puncture proof containers for sharps)
Where there are uncertainties / ambiguities around these rules? he rules themselves are fairly clear, but the uncertainties come in the form of what equipment for decontamination/inactivation is available to us as a small independent bio space which equipment such as an autoclave would be right for our space and be in accordance with city guidelines How do we work with CBC in order to insure safe disposal of waste from our lab through and to the proper facilities What are all of the ways we can implement proper safety protocol in order to meet standards and do so with finite resources and funds
Who would you work through to resolve these ambiguities/uncertanties? The first steps would be going over these items with the rest of the lab team and separating out action items and points of contact from the HTGAA program and guidelines We should as a team establish an Institutional Biosafety Committee equivalent so we can meet guidelines to dispose of waste offsite as we likely won’t have the ability to do so onsite The CBC and city departments can provide us with insight into how to comply with standards and effectively dispose of our materials off site Learning about plumbing codes and installing an anti - backflow device to our sink We can use resources in the NIH guidelines and in the area to acquire proper tagging/labelling materials and bio waste receptacles

5. Chemically hazardous waste disposal

  1. Beyond Chemical Waste and into General EH&S: Environmental Health & Safety.
    1. Most university has specialized departments that deal with member safety training, emergency procedures, hazardous waste management and disposal.
    2. Industry entities and companies often outsource these requirements to private businesses that provide EH&S services (i.e. Triume Virate, etc).
    3. OPEN QUESTIONS: What can a community lab do?
      1. Outsource? Find financial support?
      2. Form partnerships and support structures with EH&S providers?
      3. Leverage local public universities?
      4. Can partnership with city government help?
  2. Identify, Classify, Categorize Waste Types:
    1. Radioactive Waste
    2. Biohazardous Waste
    3. Mixed Waste
    4. Chemical Waste
      1. Setting up disposal zones via chemical waste types.
        1. Solids
        2. Liquids
        3. Sharps
      2. Labeling Standards & Inventory.
        1. [GHS] Globally Harmonized System of Classification and Labelling of Chemicals.
          1. Physical Hazards.
          2. Health Hazards
          3. Environmental Hazards
          4. Classification of Mixtures
        2. [NFPA 704] National Fire Protection Association’s fire diamond.
          1. Flammability.
          2. Health
          3. Reactivity
          4. Special Notice
      3. Safety Training for Members.
      4. Ongoing & Working Resources [MSDS] Materials Safety Data Sheet.
      5. Waste Disposal & Handling Supplies.
  3. Register with Dept. of Environmental Protection for a waste permit/license.
    1. Determine your lab’s waste generation capacity based on amount of chemicals you store and generate.
      • [LQG] Large quantity generator
        • > than 1,000 kg (2200 lbs.)/month of hazardous waste
        • > than 1 kg /month of acutely hazardous waste (Mass. regulations 310 CMR 30.136)
        • waste must be shipped within 90 days
        • No limit on accumulation amount
      • [SQG] Small quantity generator
        • < than l,000 kg/month
        • < than 1 kg/month of acutely hazardous
        • waste must be shipped within 180 days
        • accumulation is limited to 6000 kg in tanks and containers
      • [VSQG] Very Small quantity generator
        • < than 100 kg/month
        • no acutely hazardous waste
        • No time limit
        • accumulates MAX 1,000 kg at any time
    2. GET [QG ID #] for your lab: Self-Assigning a Hazardous Waste Generator Identification Number
    3. WHO? Either find an outside contractor or register someone in the group as a ‘contractor’ where we self-transport our waste.
    4. WHERE? Take our waste to a [TSDF] licensed treatment, storage and disposal facility or to another registered generator who is willing to take our waste.

6. Company policies for purchasing

What rules / standards locally, nationally and internationally apply?

All 6 companies we surveyed requires customer to set up an account before purchasing. On top of that, different product/item may have different regulation, such as order quantity, purpose of use. When ordering chemical/biological materials, MTA is required to be signed.

Where the are uncertainties / ambiguities around these rules?
  1. It is still hard to find out online which products are regulated and which are not. One needs to talk to the specialist in the company to figure out. It also seems that the regulation varies from company to company.
  2. It seems that companies usually supply their products to two types of customer: research institution and commercial company. There is no corresponding rules for community bio lab setup. How can one set up an account without any research affiliation?
  3. Moreover, if something goes wrong, who is gonna be responsible for that, since there is no official institution included.
Who you would work with to resolve these ambiguities / uncertainties?
  1. the best way is to talk to a company specialist.
  2. working together with major companies to establish regulations for community bio lab.
Information collected for major companies:
  1. IDT: DNA synthesis
  2. It is restricted for research use. For any purchase, the following information has be be provided:
    • Your name, address, and phone number
    • The name of your institution
    • Payment information for the order
    • The name of your Principal Investigator (PI)
    • The scale and sequence of the oligos you want to order, one oligo per line
    More details are written here about their regulation: https://www.idtdna.com/pages/home/general-information/usage-warranty-and-licenses “Research Purposes Only: Oligonucleotides and nucleic acid products ("IDT Products") are manufactured and sold by Integrated DNA Technologies, Inc. ("IDT") for the purchaser's research purposes only.”
  3. VWR: consumables
  4. It is possible to order items without an approved account. However, one can order no more than $1000 item. No chemical product is allowed to be ordered. Once an account is set up and approved, the limitation goes away. information needed to set up an account is similar to ATCC. It is not restricted to research institution.
  5. Sigma-Aldrich: chemical reagents
  6. Customer may place an order using a purchase order or a credit card. If a product requires further documentation, the Screening department will contact the customer.
  7. ATCC: cell lines
  8. They have MTA (Material Transfer Agreement) available online. One must sign before purchasing. It is prohibited to use ATCC materials for commercial use. Before purchasing, one must setup an account and wait for approval. To meet the need of commercial usage, ATCC has set up an out-licensing program that allows institutions to use ATCC products for commercial purposes under a non-exclusive license agreement. While ATCC views the Material Transfer Agreement (MTA) as an essential part of distributing its biological materials, the licensing program for commercial use of ATCC materials helps protect the ability of ATCC to supply research materials for the benefit of the scientific community.

    The detail of the MTA can be found here: In terms of distribution, ATCC ships materials direct to all countries except those restricted by the U.S. government or those who have an official ATCC distributor. Anyone ordering from ATCC or a distributor must have an approved, current account. Potential customers must be able to demonstrate that their expertise and their institution's facilities are appropriate for handling biological materials.

  9. Life technologies: bio reagents
  10. Conditions and restrictions are highly dependent on product. Generally one needs to setup an account and wait for approval before purchasing any product. It is possible to use some of their products for commercial purpose. But there is no official document pointing out which are included.

  11. Addgene: plasmid
  12. Similar to ATCC, the ordering process at Addgene follows this procedure: setup an account, get approval, select product, sign the MTA, submit the order.

    Ordering through the website is performed using these steps:

    1. Login to your Addgene account. If you need to create an account, you can register here.
    2. Find the plasmids you need in our online catalog and add them to your cart.
    3. Use our simple checkout process to complete your order online. You will need to enter payment information and billing and shipping addresses during checkout.
    4. After your order is placed, Addgene will email the required Material Transfer Agreements(MTAs) to you or your institution's Technology Transfer Office. Check out a video tutorial of our MTA process here.
    5. Once Addgene receives and approves the signed MTAs, your order will be processed and shipped.

    Background checks of customers is not required, and the institution itself does not need to officially register with Addgene. This is because each Addgene account that someone creates is affiliated with their institution. During the account creation process, each user selects the institution they are at, and this allows the MTA process described in steps 4 and 5 to move forward appropriately.