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, into large volumes of tailings water. Our system entails a certain amount of risk that is necessitated by its deployment into the field.

Given these requirements, there is an inherent risk that our synthetic bacteria might escape from their containment vessels. While the genetic systems we plan to implement do not have any direct evidence to suggest they would damage the environment, the implications of horizontal gene transfer to native microorganisms in tailings ponds and the surrounding environment is a potential issue that must be addressed. This issue has been voiced by numerous leaders in the oil industry as well as individuals living near tailing ponds. To address these concerns, we have engineered mechanical and biological safety measures that function to contain genetic elements of our synthetic bacteria. By integrating these controls, we have taken a proactive approach to the biosafety of FRED and OSCAR.

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”. Based on the suggestions outlined by Dana et al. (2012), we strongly believe that we must tackle four major safety concerns with our project. First, the synthetically engineered bacteria may be harmful physiologically 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.

Within our project design, we have worked to address all four of these concerns. 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.

Mechanical Engineered Controls

FRED

Our team has established a series of controls which we hope to implement into our biosensor during field testing and for use as a product. As above, the biosensor was designed in a completely closed system. Users will be unable to directly contact the biosensing bacterium as it is contained within sealed tubes secured with one way valves to prevent release of the organism. In field deployment of the system, the synthetic bacterium is separated from the natural environment since water samples are added through a one-directional entry port into the tube. Finally, the biosensing organism is destroyed since operators are instructed to add bleach to the one-time use sample tube once the test has been completed simply by twisting the cap of the tube.

OSCAR

The goal of the bioreactor is to be able to detoxify tailings ponds waste through the removal of nitrogen, sulfur, and carboxylic acid functional groups. The bioreactor has been designed as a separate closed system to which tailings pond water must be added rather than to add our bacterium in 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 microbes in the bioreactor vessel to escape by adhering to the belt. To counter this risk, the belt is treated with UV radiation as it exits the bioreactor solution. This process will eliminate bacteria on the belt while simultaneously maintaining integrity of the generated hydrocarbons. After this the extracted solution is sent through fractional distillation, a process which heats to over 400°C, killing any bacteria that may have gotten to this stage.

Biological Engineered Controls

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

To ensure that synthetic genetic elements do not enter the environment outside of the bioreactor and biosensor, we engineered novel biological killswitch circuits for these organisms 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 in the bioreactor and biosensor the kill genes can be suppressed. Should bacteria escape to other environments, the lack of the unique suppression conditions enables the kill system to become active.

Activation of the kill system causes the engineered cell to produce micrococcal nuclease and CviAII restriction enzyme. Our kill mechanisms are superior to previous nuclease-based killswitches because they improve completeness of DNA degradation. CviAII and micrococcal nuclease work in tandem: the endonuclease CviAII creates DNA double strand breaks at multiple sites; 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 of our bacteria escaping from the bioreactor or the biosensor.

Laboratory Personal Safety

All of the students working with the Calgary iGEM 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 of the following: authorized senior members, lab coordinators, teaching assistants, and professors.

The bacterial strains (Nocardia, Rhodoccocus, Pseudomonas, and Escherichia) used in the research are lab strains that are Biosafety Level 1 and do not pose a health risk to laboratory workers, the general public, or the environment. The team practiced appropriate procedures for working with and the disposal of tailings pond samples. Appropriate handling measures were also applied for genetically modified bacteria and materials contaminated with bacteria. The measures followed were according to the MSDS and the biosafety regulations present at the University of Calgary. By following 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 all non-pathogenic in addition to E. coli.

References

Dana G, Kuiken T, Rejeski D & Snow A (2012) Synthetic biology: Four steps to avoid a synthetic-biology disaster. Nature 483: 29.