The iGEM Safety Form asks us as students to think about our projects in a biosafety context. What would happen if our bacteria were to escape from the lab? How would species, ecosystems, or even individuals be affected by the genetically engineered microorganisms (GEM) we are creating? These are incredibly important questions that need to be answered in order to ensure a safe and well-protected laboratory environment.

If a GEM is accidentally or purposefully released from a lab, there may potentially be devastating consequences. The fear is that bacteria will escape and colonize outside of the lab, replicating beyond human control. Once colonized, GEMs may become endemic to that area, upsetting the balance of natural flora by either out-competing species to extinction, directly damaging species, or potentially harming the ecosystem itself. As described by Tucker and Zilinskas:

“If a synthetic microorganism is built by combining these genetic elements in a new way, it will lack a clear genetic pedigree and could have “emergent properties” arising from the complex interactions of its constituent genes...including its possible spread into new ecological niches and the evolution of novel and potentially harmful characteristics.” (Tucker et. al, 2006)

Despite the fact that GEMs are generally considered less fit than their wild-type counterparts, the chance of colonization outside the lab is still one which is prevalent in the minds of concerned biologists, the general public, as well as “watchdog organizations” such as GeneWatch UK. While there have been no reported GEM outbreaks in the past, synthetic biology is a rising field of study and for the sake of the public safety, as well as the image of synthetic biology as a helpful and important scientific discipline, the time to ensure procedures are safe is now.

We believe that our kill switch tool could be utilized to better ensure that a widespread outbreak of GEMs does not occur. Post-release, a GEM would need to find a food source and an environment in which it can replicate and grow viably. Inserting our kill switch tool into bacteria would serve as an additional safety net if the GEM were to be released by prospectively predicting a unique feature of its extra-laboratorial environment and engineering a promoter to activate intracellular AMP production selectively in the presence of this chemical, causing the bacteria to subsequently die and thus severely reducing the chance of colonization.

The beauty of this is that the idea is highly portable to many safety applications. The way in which the kill switch is induced can be tailored depending on the system it is to be implemented in. Intracellular AMP production could be induced by as specific or as broad a niche as fits the parameters of the experiment. Obviously, too specific a niche reduces the chances of the anti-colonization effect occurring, and too broad a niche may lead to accidental cell death, but there is certainly a wide breadth in between those two scenarios. By changing the promoter attached to the protegrin-1 gene, what induces AMP production could be entirely up to whatever safety committee would be in charge of lab security. This could range from contact with chemicals in local water supplies, certain foodstuffs, even specific species of animal, including, but not limited to, humans. These details could be based off the specifics of the bacteria in question (including its preferred ecological niche, known intracellular chemical pathways etc.) or the environment into which it may colonize (such as various chemical stimuli, food the bacteria may encounter, temperature, pH, etc.), creating a metaphorical safety net which could be effectively deployed in addition to existing bio-safety methods. The possibility would also exist to use a negatively-induced promoter to activate the kill switch. This would involve using a synthetic chemical that is not present in the bacteria's ecological niche to inactive the kill switch promoter, allowing the cultures grow uninhibited within the lab. If the organism were ever to escape the lab, it would loose its source of this chemical, causing intracellular production of AMP to be activated, resulting in cell death. This option may well be easier than having to engineer a new, positively regulated promoter, as one could use a standard, or set of standard, molecules to ensure viable conditions only exist within the laboratory.

Not only would it be effective, the kill switch is simple to implement as well. Inserting the AMP gene is as easy as ligating it to a plasmid backbone and if needed, it can later be removed via miniprepping the plasmid and digesting out the gene. Subsequent replication of these cells would allow for an entire colony of GEMs to have an inducible kill switch that would ensure colonization outside the lab would not occur. Our lab was able to ligate and transform the gene into E. coli. The protegrin-1 coding region is only 54 base pairs long, making it cheap, affordable, and able to be readily synthesized by DNA laboratory with appropriate facilities. Also, the 18-residue protein is small enough that it will fold in vivo without the requirement of chaperone proteins (Jang, 2007).

As synthetic biology becomes a greater and greater part of the scientific limelight, biosafety will become an important issue to address in both the minds of fellow scientists, as well as the public at large. The risk of GEM release from a laboratory is not a threat to be taken lightly, and could have a devastating impact on the condition and viability of environments, populations of flora (both uni and multicellular in nature), and even humans. Our kill switch will provide scientists the ability to safely work with microbes, knowing that the risk of colonization from any accidental or purposeful release will be significantly reduced by the activation of intracellular AMP production. We foresee the use of this regulatory technique alongside many other standard biosafety protocols in order to promote the safest work environment possible.

References:

Tucker, Jonathan and Zilinskas, Raymond. “The Promises and Perils of Synthetic Biology”. The New Atlantis, Number 12, Spring 2006. pgs. 25-45. Link to paper.

Jang, Hyunbum et al. "Conformational Study of the Protegrin-1 Dimer Interaction with Lipid Bilayers and its Effect." BioMed Central Structural Biology, Vol. 7. Published April 2007. Link to paper.