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| - | <h2>Purpose:</h2> | + | <p> The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. Despite our belief that the metabolic burden of this system on our bacteria would not allow them to outcompete any native organisms, as we detail in our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">interviews</a> page, our dialogue with experts really emphasized the need to design such a system so as to minimize any escape of our bacteria regardless. As such, we designed a closed <a href=https://2012.igem.org/Team:Calgary/Project/FRED/Prototype>biosensor</a> and a closed <a href=https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor>bioreactor</a> which incorporated built-in structural <a href=https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design> design</a> safety mechanisms. In order to implement one more level of control, which industry felt was needed, we wanted an additional genetic-based containment mechanism to kill our bacteria upon escape from our system, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We implemented novel ribo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.</p> |
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| - | <p>The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. As we recognize in our project's <a href="https://2012.igem.org/Team:Calgary/Safety">safety practices</a>, remediating tailings ponds creates the risk for our bacteria to escape into the environment, given the high volume of tailings water being circulated through our system. Although no evidence suggests that the OSCAR bacteria would proliferate or cause harm outside the bioreactor, industry leaders have voiced concerns to our team about synthetic biology's potential for environmental issues—safety is different in synthetic biology from more traditional engineering disciplines because bacteria are self-replicating entities. Thus, we cannot predict all possible consequences of OSCAR escaping the bioreactor.</p>
| + | <h2>Click on either element to learn more about it!</h2> |
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| - | <p>To counter potential safety issues, we engineered a genetic-based containment mechanism into our bacteria. Our kill switch system is designed to destruct the organism's synthetic genes upon escape from the bioreactor, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We hope that our proactive approach to safety in OSCAR will promote adoption of our synthetic biology system for remediation of tailings ponds in the conventional oil sands industry.</p>
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| - | <h2>Our System:</h2> | + | |
| - | <p>Two basic elements comprise our active kill system: firstly, two nucleases are used to degrade the genome; and secondly, a regulatory platform is used to control these two kill genes. The nuclease enzymes ensure degradation of synthetic genes upon kill switch activation. Such a nuclease mechanism is superior to widely used lysis-based techniques which leave genetic material intact, allowing its release into the environment and potential horizontal gene transfer into other organisms. Our team submitted into the registry two novel <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">kill enzymes</a>. These are noteworthy because they are optimized for temperature conditions typical of tailing ponds. Furthermore, they cause finer degradation of the bacterial genome than existing nuclease mechanisms such as (SHOW ME ONE).</p>
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| - | <p>Of course, introducing nucleases is insufficient to control the spread of our organism and its genes. Our system also required a means of <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">regulating</a> the expression of these nucleases, thereby allowing the organism to function as intended while within the bioreactor environment. To fulfill this need, we developed four novel regulatory elements for registry submission, significant because they are tightly regulated—these elements reduce inadvertent expression of the kill genes compared to existing regulatory mechanisms. Three are of particular interest because they are riboswitches that regulate translation at the post-transcriptional stage of gene expression. Of the four we developed, two proved suitable for tailings pond use.</p>
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| - | <h2>Justification of our approach</h2>
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| - | <p>An apparent weakness of our system lies in the potential for the kill switch mechanism to mutate, rendering it ineffective and allowing the synthetic organism or its genetic material to escape into the environment. We have, however, taken several approaches to mitigate this risk. Firstly, we engineered redundancy into our system—with two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10^-6 per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor (LINK TO SAFETY). Only when these measures fail will the kill switch be required to function.</p>
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| - | <p>At first blush, an auxotrophic bacterial strain would seem a superior choice for our application. Such a strain would have a critical metabolic gene knocked out, requiring the organism be supplemented externally with an intermediate metabolite. As a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, an auxotrophic mechanism seems ideal. This system, however, is impractical due to its cost. Metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As we learned in our discussions with industry leaders, cost is a paramount factor in permitting adoption at industrial scales, and so our system has a greater likelihood of being implemented outside the laboratory. More worrisome is that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. As the overarching goal of our kill switch is not to kill the organism but to prevent the escape of its engineered genetic elements, our kill switch design is superior to one relying on auxotrophy.</p>
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| | + | <a style="margin-left: 20px;" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><img src="https://static.igem.org/mediawiki/2012/d/d5/UCalgary2012_FRED_Killswitch_Regulation.png"></img></a> |
| | + | <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes"><img src="https://static.igem.org/mediawiki/2012/f/fd/UCalgary2012_OSCAR_Killswitch_KillGene.png"></img></a> |
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A Killswitch for Increased Security
The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. Despite our belief that the metabolic burden of this system on our bacteria would not allow them to outcompete any native organisms, as we detail in our interviews page, our dialogue with experts really emphasized the need to design such a system so as to minimize any escape of our bacteria regardless. As such, we designed a closed biosensor and a closed bioreactor which incorporated built-in structural design safety mechanisms. In order to implement one more level of control, which industry felt was needed, we wanted an additional genetic-based containment mechanism to kill our bacteria upon escape from our system, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We implemented novel ribo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.
Click on either element to learn more about it!
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