Safety

The Risks of FRED and OSCAR

Our project utilizes two types of engineered bacteria to detoxify tailings water. To quantify the amount of the toxic compounds present in the tailings water we are relying on FRED, a biosensor bacterium, that will work inside a closed environment to detect the toxins. Within our bioreactor system, we intend to introduce OSCAR, a bacterium capable of detoxifying toxic compounds, to process large volumes of tailings water.

Whenever engineered bacteria are used, there is an inherent risk that the bacteria might escape from their containment vessels into the surrounding environment. There is no direct evidence to suggest the genetic systems we have implemented would have a negative impact on the environment. However, the implications of horizontal gene transfer to native microorganisms in tailings ponds and the surrounding environment must be addressed. This issue has been raised by numerous leaders in the oil industry as well as individuals living near tailing ponds. Our approach to biosafety was inspired by a comment published in Nature (Dana et al., 2012) which suggested multiple ways to prevent “Synthetic Biology Disaster”. We strongly believe that we must tackle four major safety concerns with our project. First, the synthetically engineered bacteria may be harmful to natural flora in the environment. Second, the bacteria may not only survive in the tailing pond environment but thrive in it, allowing it to outcompete naturally occurring bacteria. Third, genes may be transferred from our synthetic bacteria to native organisms. Fourth, if any of our genetically modified bacteria were to be able to grow in the tailings pond, evolution may allow for mutations which prevent our safety measures from working.

To address these four major safety concerns, we have engineered mechanical and biological safety measures that function to contain genetic elements of our synthetic bacteria. The first two concerns have been addressed through mechanical engineered controls by physically separating our organisms from the native environment. The third concern has been addressed through the development of a novel kill switch system to prevent our engineered organisms' DNA from spreading to other organisms. The fourth concern will be addressed by producing redundancy in our kill switch system which can be applied in the scale-up process of our project. By integrating these controls, we have taken a proactive approach to the biosafety of FRED and OSCAR.

Mechanical Engineered Controls

FRED

Our team has established a series of controls which we hope to implement in our biosensor during field testing and optimization of the final product. The final product will contain FRED within one-time use closed containers, with one-way valves. The operator will insert water samples through the one way valve, isolating FRED from the operator and the environment. Once testing is completed, the operator will be instructed to simply twist the cap of the tube to release a pre-measured aliquot of bleach to destroy FRED prior to proper disposal of the container.

OSCAR

The goal of OSCAR is to be able to detoxify tailings ponds compounds through the removal of nitrogen, sulfur, and carboxylic acid functional groups. OSCAR has been designed to function in a closed system to which tailings pond water is added, as opposed to adding OSCAR directly into the environment. Hydrocarbon products generated in the bioreactor after microbial remediation are collected from the culture with a continuous belt skimming device. There is potential for OSCAR in the bioreactor vessel to escape by adhering to the belt. To counter this risk, the belt is treated with UV irradiation as it exits the bioreactor solution. This process will destroy OSCAR on the belt while simultaneously maintaining integrity of the generated hydrocarbons. The extracted solution is then sent through fractional distillation, a process which heats the hydrocarbon solution to over 400°C, killing any OSCARs that may have survived UV exposure.

Biological Engineered Controls

Kill switches that have previously been entered into the registry often rely on methods that induce cell lysis. In these systems, genetic material is left intact, allowing for the remaining DNA to be taken up by bacteria and introducing the possibility that synthetic genes escape into the environment. We feel that lysis based kill switches are insufficient for use in FRED and OSCAR, necessitating design of novel kill switches.

To ensure that synthetic genetic elements cannot escape the closed systems in which FRED and OSCAR will be used, we engineered novel biological kill switches which we named "Ribo-Kill-Switches." These Ribo-Kill-Switches initiate cell death through degradation of genomic and plasmid DNA. Through a unique cell culture condition within the closed bioreactor and biosensor systems the kill switch genes can be suppressed. Should bacteria escape, the lack of the unique suppression conditions enables the kill switch system to become active.

Activation of the kill switch system causes the engineered cell to produce micrococcal nuclease and CviAII restriction enzyme. Our kill switch mechanisms are superior to previous nuclease-based kill switches because we have improved the completeness of DNA degradation. CviAII and micrococcal nuclease work in tandem: the endonuclease CviAII creates DNA double strand breaks at multiple sites while the micrococcal exonuclease activity degrades remaining strands into single nucleotides. The degradative enzymes chosen for our system were specifically selected for their ability to function at low temperatures, in variable pH conditions, and to work quickly to degrade as much of the genetic material as possible. These engineered biological controls ensure that synthetic genetic elements are completely destroyed in the event that FRED or OSCAR escape from the closed bioreactor or biosensor systems.

Laboratory Personal Safety

All of the students working with the iGEM 2012 Calgary Team received appropriate safety training as described by the University of Calgary’s safety policies. This included a Biosafety course which introduced the students to proper handling of biological materials. In addition, all iGEM students were required to attend proper Workplace Hazardous Materials Information System (WHMIS) training sessions. All safety procedures and guidelines of “Level 2 Biohazard Labs” were followed. Students were also supervised at all times by at least one authorized senior member, lab coordinator, teaching assistant, or professor.

The bacterial strains (Nocardia, Rhodoccocus, Pseudomonas, and Escherichia) used in the research are lab strains rated as Biosafety Level 1 and do not pose a health risk to laboratory workers, the general public, or the environment. The team also practiced appropriate procedures for working with and the disposal of tailings water samples. Appropriate handling measures were also applied for genetically modified bacteria and materials contaminated with bacteria. All measures outlined in the Material Safety Data Sheets (MSDS) and the biosafety regulations present at the University of Calgary were followed.

Through these procedures, none of the genetically modified bacteria could have a chance of being introduced into the environment. The constructs that we have built to test our systems in the laboratory all used a safe, non-pathogenic bacterial strain of E. coli commonly used in labs worldwide. Other bacteria which were used for characterization of their genes, as listed above, are also non-pathogenic.