Safety

The Risks of FRED and OSCAR

The very functions of our project require our engineered bacteria to be in close proximity to the outside environment. With the bioreactor, we intend to maximize removal of toxins from tailings water requiring the OSCAR bacterium to be present in large volumes of industrial wastewater. While quantification of toxins with our biosensor FRED will take place in a closed environment, there is risk necessitated with field deployment.

Given these parameters, 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 other environments is a potential issue which must be addressed. This issue has been voiced by numerous leaders in the oil industry as well as individuals living near areas where tailing ponds exist. Therefore we engineered mechanical and biological safety measures which function to contain genetic elements of our synthetic bacteria. By integrating these controls, we have taken a proactive approach to biosafety of FRED and OSCAR.

This work was inspired by a Comment published in Nature (Dana et al. 2012) which suggested multiple ways to prevent “Synthetic Biology Disaster”. By applying this to our project we strongly believe that we must tackle four major safety components of our project. Firstly the synthetically engineered bacteria may be harmful physiologically to natural flora in the environment. Secondly, the bacteria may not only survive in the tailing pond environment but thrive in it allowing it to outcompete natural bacteria. If any genetically modified bacteria were to be able to grow in the tailing pond, evolution may allow for mutations which prevent safety measurements from working. Finally, genes may be transferred from the synthetic organism to other native organisms. We have addressed the first two concerns through mechanical engineered controls, separating our organism from the environment. The fourth concern has been addressed through development of a novel kill switch system to prevent DNA spread to other organisms. The third concern can easily be addressed by producing redundancy in our kill switch system which we hope to be able to apply in the scale up process of our project.

Mechanical Engineered Controls

OSCAR

The goal of the bioreactor is to be able to detoxify tailings ponds waste through the removal of nitrogen, sulfur, and carboxylic acid moieties. As such, 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.

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.

Biological Engineered Controls

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 devices 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 the bacteria escape to other environments and the lack of suppression enables the kill system to become active. The degradative enzymes chosen for our system were especially selected for their ability to function at low temperatures, in variable conditions, and to work quickly to degrade as much of the genetic material as possible.

Although there are numerous kill switch systems that in the 2011 Parts Registry, these circuits were insufficient for the use in FRED and OSCAR, which necessitated us to design novel kill switches. Kill switches previously entered into the registry typically kill bacteria through some sort of lysis-based mechanism. Although such switches can effectively kill cells, genomic material is left intact, and thus synthetic gene constructs would be permitted to spread into the environment.

To counter this risk, our kill system is based on enzymatic degradation of the bacterial genome. Activation of the kill system causes the engineered cell to produce micrococcal nuclease and the CviAII restriction enzyme. As opposed to other enzymatic kill system in the 2011 Registry, our kill mechanisms are superior because they improve completeness of genomic degradation. CviAII and micrococcal nuclease work in tandem: firstly, by breaking up the genome into manageable chunks; and secondly, degrading these chunks into single nucleotides. 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 policy. This included a Biosafety course which introduced how to properly handle biological materials. In addition 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 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 protocols, none of the genetically modified bacteria have a chance to be introduced into the environment. The constructs which have been built to test our systems in the laboratory all use 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.