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Team:Calgary/Project/HumanPractices/Killswitch
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A Killswitch for Increased Security
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Purpose:
Synthetic biology entails designing an organism to do a specific task. This involves genetic manipulation and requires scientists to provide the bacteria with a selective advantage such as an antibiotic cassette which forces the bacteria to keep the gene of interest inside the cell. With such manipulation comes a valid “risk of accidental release” (Tucker and Zilinkas, 2006). In order to prevent such a bacteria from becoming rogue a killswitch is designed such that the bacteria is only able to survive in specific environments allowing them to perform the tasks of decarboxylation, denitrification and desulfurization in our bioreactor. However, in case of these bacteria escaping, the lack of a metabolite and or the presence of a particular metabolite will activate the “kill genes” which will cause the bacteria to self destruct. The killswitch mechanism was put in our system as a safety measure in addition to the bioreactor to contain the synthetic bacteria.
History:
Scientists have been trying to develop methods to limit bacterial viability and growth outside of the lab environment. One of the most popular methods used to ensure the safety of bacteria used in the lab was the creation of lab strain bacteria such as DH5α and Top10. These bacteria are metabolically deficient and are unable to survive outside of the lab environment without very specific nutrients. Additionally, The Registry of Biological Parts also has several killswitches readily available that were submitted by previous iGEM teams.
The different types of killswitches include:
Inducible kill genes: Inducible systems generally consist of a regulatory element such as a promoter which is activated in the presence or absence of a metabolite. GIVE EXAMPLES
Toxin-antitoxin systems: These systems usually insert antitoxin in the plasmid and toxin in the genome. Ideally if the bacteria lose the plasmid then the bacteria dies. This does not address the problem of horizontal gene transfer between species.
Auxotrophic marker
Design considerations:
<p> During the first phase of design we considered classic systems such as auxotrophic markers, toxin-antitoxin systems, inducible systems. However, considering the cost of the system if auxotrophic markers were used we did not pursue that route. We have decided to use the inducible systems. We explored four different inducible systems which are induced by inexpensive ligands such as magnesium, manganese, molybate salts and glucose. In order to make sure the systems are controlled well and the kill switch regulation is not leaky, we have added an additional control using the riboswitch.A riboswitch provides post-transcriptional control of gene expression. A riboswitch is a small stretch of mRNA which binds to a ligand which increases or decreases the expression of the gene downstream.
