Safety on Our Project: Endocrine Disruptor Bioremediation

Researcher Safety

A hazardous waste tag on endocrine disruptor liquid waste

While most of our project is in lab strains of E. coli, (NIH Risk Group 1) we are culturing organisms from environmental samples that are able to survive in minimal media containing one of the four endocrine disrupting compounds (EDCs) we are hoping to degrade. Past studies have found Pseudomonas species (Kang, Katayama et al. 2006) and Sphingomonas species (Sasaki, Maki et al. 2005) capable of degrading bisphenol A. These genera can be found in NIH Risk Groups 2 and 3. Due to immediately culturing in minimal media, it is unlikely that we will culture any significantly pathogenic organisms from the LA River, according to the Caltech Safety Office.

We are working with endocrine disrupting compounds. These affect the endocrine system by competing with hormones to bind nuclear receptors, and can act as agonists or antagonists affecting the transcription of the many genes controlled by the presence or absence of hormones. We have chosen to focus on those that can bind to the estrogen receptor, as these particular endocrine disruptors have been shown to cause higher prevalence of intersex fish near sources of EDC pollution (Wise, O'Brien et al. 2011). The four chemicals we are focusing on are: bisphenol A, nonylphenol, 17a-ethynylestradiol, and 4,4’-DDT.

These EDCs are harmful to human health. They affect the reproductive system by their nature as endocrine disruptors. 4,4' DDT and 17a-ethynylestradiol are known carcinogens. 17a-ethynylestradiol is a known teratogen (MSDS). We take care to use small amounts and avoid exposure by contact or inhalation.

The screen described in Kiyohara et al., 1982, which requires the water-insoluble EDCs to be dissolved in a volatile solvent and sprayed on plates, was deemed too unsafe for our lab. The risk of potential researcher and environmental exposure due to spraying chemicals, even with secondary containment was deemed too great, since we do not have any permanent setup for such a procedure. The safety office was also concerned that if we were able to create satisfactory secondary containment for the vapor, we would create an explosion hazard from the high concentration of acetone in an enclosed space. The safety office recommended an alternative where we pour endocrine disruptors dissolved in acetone on plates rather than spray to minimize inhalation risk, using an orbital shaker to attempt to create an even coat. We used this method to add ECDs to minimal media agar plates in the hood. The researcher doing this procedure wore a lab coat and safety glasses at all times to minimize exposure to the EDC-acetone solutions.

Public Safety

Once our bioremediation system can be used in the field, most safety concerns to the public are similar to researcher or environment ones. The sites selected for remediation would likely contain concentrated endocrine disrupting compounds and the health risks would be the same as for the researchers. These areas should have limited access before and during remediation to prevent human exposure to EDCs and the genetically engineered organisms for degradation. Containment systems for the engineered microbes are discussed below in Environmental Safety.

Environmental Safety

We envision our bioremediation system being used in an aquatic setting such as a river or aquifer rather than a terrestrial one. Different considerations must be made for physical containment when working with a liquid versus a solid environment.

These EDCs are particularly toxic to aquatic life. We avoid allowing any amount of these chemicals to go down the drain and instead collect waste in a hazardous waste container as described by the Institute’s hazardous waste policies.

For field use, our genes must be placed in a chassis that will have direct contact with polluted water. We must try to minimize environmental contamination of both the organism and its recombinant DNA as per NIH guidelines.

One common method of in situ bioremediation of ground water, using microorganisms present at the site, is to have wells. One is used to inject a carbon source and electron donor, and a later well is where purified water can be found and tested (Hwang, Wu et al. 2009). This injection allows humans to increase the rate of degradation by the microbe community at the site. This sort of system is invasive and would be best used in sites that require long-term remediation, such as the uranium site in Hwang et al.

Because of NIH and EPA guidelines, there have been few field trials for bioremediation using genetically modified organisms. Then first approval of use of genetically modified bacteria in the field was in 2000, by Ripp and al. They used Pseudomonas fluorescens HK44 to degrade naphthalene. It also contained lux, which allowed for visual tracking of the genetically modified organisms (Urgun-Demirtas, Stark et al. 2006). For genetically modified bioremediation to get out of labs and into the field, we need to have some sort of containment system.

There are few suitable containment systems currently available for the proposed application of our project. Most membrane bioreactors, primarily used in wastewater treatment plants, are relatively large and would require a permanent structure. They need a pump to force the water to flow through the membrane and constant cleaning to prevent build-up on the membrane. However, these systems are effective at separating microorganisms and solids from clean effluent by having pores in the cellulose membrane smaller than most bacteria (1 micron) (EPA Fact Sheet). This would physically prevent the genetically modified organisms from being released into the environment. Any native flora, fauna or microbe would also be stuck in the bioreactor. A selective inlet would prevent most large objects and organisms from entering the bioreactor while still allowing EDC-contaminated water in. The physical containment could fail if any large debris pierces the membrane, so this could not be the only method of containment (EPA Fact Sheet).

A more permanent physical bioreactor would be of more use in a dedicated Superfund site, such as Montrose Chemical Corp in LA County. This factory manufactured DDT from 1947 until 1982, contaminating the groundwater and soil near the site with DDT and chemicals required to make DDT. These sites would have much more concentrated amounts of pollutants in the soil and water, making it easy the bioremediation system to obtain pollutants to degrade. However, we envision a more portable bioremediation system, as sites of EDC spills vary widely. Runoff from farms (a major contributor of synthetic and natural estrogens to water) would be an instance of needing a portable system (Wise, O'Brien et al. 2011).

Another method of containing genetically modified bacteria is through biology. These prevent recombinant plasmids from being taken up by native species (horizontal gene transfer) and to prevent the engineered strain from outcompeting the local community. A common method is to use the ccdB gene ([http://partsregistry.org/Part:BBa_P1016 BBa_P1016] and [http://partsregistry.org/Part:BBa_P1010 BBa_P1010])on a plasmid in conjunction with an E. coli strain such as DB3.1 ([http://partsregistry.org/Part:BBa_V1005 BBa_V1005]). The plasmid will code for the “death gene” which will kill any cell that does not code for immunity in its genome. If a native microorganism would uptake this manmade plasmid, it would die, preventing the propagation of the recombinant DNA in the environment (Featured Parts: Cell Death).

Another similar “suicide” containment system uses streptavidin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J36841 BBa_J36841]) (Kaplan, Mello et al. 1999; Urgun-Demirtas, Stark et al. 2006). This protein binds very tightly to biotin, a required co-enzyme for many metabolic pathways. This makes biotin unavailable and causes cell death. Kaplan et al. reported cell counts were reduced 99.9% in eight hours after their system was activated by absence of pollutant to degrade.

Normal lab strains are engineered so that they will be unable to survive in the field. A more durable chassis will be needed such as Pseudomonas species or Deinococcus radiodurans (Urgun-Demirtas, Stark et al. 2006). Their durability makes them more difficult to contain, but the cost of attempting to keep E. coli alive in field use may be too high. To be able to use E. coli, one might need to inject nutrients into the bioreactor, a possibly expensive process, especially if done over a long period. Depending on the physical containment system, these injected nutrients might be able to escape into the ecosystem, potentially causing blooms of some species and destroying the balance of the microbial community. Physically cycling E. coli in the reactor would replace dead cells with live ones quickly, but this might also be prohibitively expensive.

New BioBricks

Our new biobricks are able to degrade BPA or DDT. The p450, WT-F87A is well characterized for many reactions, as it is promiscuous. We are analyzing its degradation of bisphenol A using HPLC. The DDT dehydrochlorinase is relatively unknown. Neither enzyme is expected to be hazardous, however, the promiscuity of WT-F87A may cause unwanted reactions with other organic molecules.

We will determine the metabolites of a degradation pathway using HPLC and mass spectroscopy. We can then look in the literature for any evidence of toxicity or estrogenicity, as there is a possibility that the metabolites are worse environmental toxins than the endocrine disruptors themselves.

Biosafety Regulations

Puikei showing off our liquid hazardous waste and biowaste containment area.

International:

• [http://www.who.int/csr/delibepidemics/WHO_CDS_CSR_LYO_2004_11/en/ WHO]
• [http://bch.cbd.int/protocol/ Convention on Biological Diversity]

National:

• [http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm NIH]
• [http://www.cdc.gov/biosafety/ Center for Disease Control (CDC)]
• [http://www.absa.org/ American Biological Safety Association]
• [http://www.epa.gov/opptintr/biotech/index.htm US Environmental Protection Agency (EPA)]

Local:

• [http://safety.caltech.edu/ Caltech Safety Office]
• [http://safety.caltech.edu/committees/committees_biosafety Caltech Institute Biosafety Committee]
  • [http://safety.caltech.edu/documents/24-biosafety_manual.pdf Caltech Biosafety Manual (pdf)]
  • [http://safety.caltech.edu/documents/22-biohazardous_agent_classification.pdf Caltech Biohazardous Agent Classification (pdf)]
    
    

We talked to the safety office about our project. They saw no problems with our handling of biological safety since we are working with Level 1 organisms. They noted that the possibility of growing pathological bacteria from our enrichment cultures is low due to immediately diluting in minimal media rather than growing environmental samples up on LB. If we suspect any of the cultured organisms are harmful, we will work with the safety office to implement proper safety procedures.

However, we talked to the safety office about some of the possible chemical hazards in our lab. They directed us to dispose of endocrine disrupting compounds as hazardous waste and taught us how to properly label and store the container. We were also instructed to make sure anything containing EDCs is stored in secondary containment. Any solid waste (tubes, etc) should be put in a container or bag and also labeled as hazardous waste. We will place containers containing EDCs in secondary containment as much as possible to avoid researcher exposure or environmental contamination.

The team completed safety training including a walkthrough the lab with the lab safety officer before beginning any lab work. We also spent about 3 days with the graduate student mentors learning molecular biology techniques including how do be safe while doing so.

How we comply with Institute Guidelines

Training

Before working in the lab, we were required to attend the SURF Safety Presentation, a talk given by the Caltech Safety Office that goes over general guidelines for working in any sort of lab.

• How to assemble if there is a fire
  • For Braun, outside by Wilson Ave, letting the floor warden know if everyone from your lab evacuated
• Shelter in place for earthquakes and other hazards
  • Emails, phone calls and texts through the automated message system will alert everyone to threats on campus
• Wear appropriate attire to work
  • Closed toed shoes
  • Long pants if working with large amounts of dangerous chemicals
• Avoid working alone and let someone know that you're in lab if you must work alone
• For emergencies, call security at ext 5000.

The safety officers for Braun 16 are Dr. Michael Vicic, Linda Song and Grayson Chadwick. They gave us a tour of the lab, showing us were safety equipment was before getting card access to the building and lab.

• Shower and eyewash located near the main door
• Fire extinguisher near main exit
• First aid kit at front of room
• Telephone near main exit

Our graduate student mentors, Emzo de los Santos, Joe Meyerowitz, Nate Glasser and Toni Lee gave us a 2-day bootcamp before we could begin work on the endocrine disruptor project. This included how to perform sterile technique, operate the equipment we would need in the lab, and basic molecular biology techniques, since 4 team members had no previous research experience.

Concise Guidelines for iGEM Researchers

(See above in Researcher Safety for a more detailed description of endocrine disruptor hazards)

• When handling cells, DNA or chemicals, researchers are required to wear gloves and change them often. This protects us and our experiments.
• When handling endocrine disrupting compounds, researchers must wear, in addition to gloves, a lab coat and safety glasses, particularly when working with EDCs dissolved in acetone.
  • Work with EDCs must be done in the chemical hood if possible.
  • Researchers should use the smallest amount possible of an EDC in experiments to avoid waste, because trace amounts have been enough to cause harm to aquatic life.
• DNA in agarose gels are stained with Sybr Safe instead of ethidium bromide.
  • Gel imaging is still done under UV, so we minimize researcher exposure by
    • Making sure the image box is closed before turning the UV light on, and making sure the UV light is off before opening it.
    • Performing gel extractions with a blue light box, which only requires amber glasses

2011 Caltech iGEM's general procedure to comply with Institute requirements for disposing of biowaste and hazardous waste

Ideas for General iGEM Safety

We believe requiring more documentation of submitted parts will help improve the safety of iGEM teams. Having more information about what we are working with allows us to design better experiments, understand interactions between parts and be aware of possible dangers to ourselves or the environment caused by the BioBrick. Some ways we think teams can help make their documentation even better:

• Cite literature used in the creation of the BioBrick
• Provide documentation of the creation and use of the part by writing directly on the Part page or linking to the team's notebook or project page
• If possible, sequence the part right before submitting it to the Parts Registry. Use that sequence rather than the intended or planned sequence in the part specification. If it is different from the intended sequence, note that on the Part Design or the BioBrick's main page.
• Fill out the experience page with data showing how the part worked for a specific test or link to the team wiki about use of that part. Experience pages are useful for helping teams decide what part to use if there are multiple choices to fulfill their need for their project.

References

• 4,4' DDT; MSDS No. 386340; Sigma-Aldrich: St. Louis, MO, April 8, 2011.
  
• 17a-ethynylestradiol; MSDS No. E4876; Sigma-Aldrich: St. Louis, MO, Jan 18, 2011.
  
• Bisphenol A; MSDS No. 133027; Sigma-Aldrich: St. Louis, MO, March 13, 2010.
  
• EPA. "Montrose Chemical Corp." Region 9: Superfund. Revised Feb 1, 2011. Accessed July 13, 2011. [http://yosemite.epa.gov/r9/sfund/r9sfdocw.nsf/vwsoalphabetic/Montrose+Chemical+Corp?OpenDocument http://yosemite.epa.gov/r9/sfund/r9sfdocw.nsf/vwsoalphabetic/Montrose+Chemical+Corp?OpenDocument]
• EPA. Wastewater Management Fact Sheet: Membrane Bioreactors. [http://water.epa.gov/scitech/wastetech/upload/2008_01_23_mtb_etfs_membrane-bioreactors.pdf http://water.epa.gov/scitech/wastetech/upload/2008_01_23_mtb_etfs_membrane-bioreactors.pdf]
  
• Hazardous Waste Management Reference Guide. California Institute of Technology Environment, Health and Safety Office
  
• Hwang, C., W. Wu, et al. (2009). "Bacterial community succession during in situ uranium bioremediation: spatial similarities along controlled flow paths." The ISME journal 3(1): 47-64
  
• Kaplan, D. L., C. Mello, et al. (1999). "Streptavidin-based Containment Systems for Genetically Engineered Microorganisms." Biomolecular Engineering 16: 135-140.
• Kang, J. H., Y. Katayama, et al. (2006). "Biodegradation or metabolism of bisphenol A: from microorganisms to mammals." Toxicology 217(2-3): 81-90.
  
• NIH Guidelines For Research Involving Recombinant DNA Molecules. Revised May 2011. Accessed July 11, 2011. [http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm#_Toc7261555 http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm#_Toc7261555]
• Nonylphenol; MSDS No. 290858; Sigma-Aldrich: St. Louis, MO, July 21, 2010.
  
• "Feature Parts: Cell Death." Registry of Standard Biological Parts. Revised March 16, 2010. Accessed July 13, 2011. [http://partsregistry.org/Featured_Parts:Cell_Death http://partsregistry.org/Featured_Parts:Cell_Death]
• Sasaki, M., J.-i. Maki, et al. (2005). "Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1." Biodegradation 16: 449-459.
  
• Urgun-Demirtas, M., B. Stark, et al. (2006). "Use of genetically engineered microorganisms (GEMs) for the bioremediation of contaminants." Critical reviews in biotechnology 26(3): 145-164.
  
• Wise, A., K. O'Brien, et al. (2011). "Are Oral Contraceptives a Significant Contributor to the Estrogenicity of Drinking Water?" Environmental Science and Technology 45(1): 51-60.