Synthetic bugs on the loose: containment options for deeply engineered (micro)organisms
Graphical abstract
Introduction
Since the birth of modern Genetic Engineering in the mid-70s of the past century there has been a recurrent concern that live entities deliberately modified for acquiring non-natural properties could cause unexpected effects when released into the environment. The arch-famous Asilomar conference of 1975 [1, 2] much echoed these concerns, which by that time were limited to the possible accidental escape of the microbes at stake [3]. One decade later, the pioneering work of Ananda Chakrabarty [4] and Kenneth Timmis [5] entered yet one more screw turn to the subject by proposing the deliberate release of genetically engineered (GE) microorganisms (GEMs) for bioremediation of sites polluted extensively with chemical waste. In this case, the challenge was not to contain these agents by making them weaker (as was the main outcome of Asilomar), but to spread them and ensure delivery of the expected activities where and when needed, while avoiding their proliferation beyond the target spatiotemporal scenario [6]. These two main concerns (preventing accidental escape and containing any unplanned dispersion or survival of the GEMs beyond a given time and place) translated along the years into different propositions. These included both barriers to horizontal gene transfer of recombinant DNA and conditional survival of the engineered agents limited only to precise specifications. The many strategies to these ends have been reviewed a number of times [7••, 8••]. In reality none of such earlier containment attempts granted an expected escape frequency that was low enough to be acceptable, but pointing in the way a number of bottlenecks that needed to be tackled for making active or passive containment a reality. In the meantime the lack of any serious health or environmental incidents involving GEMs for the next 20 years made the field to go into some sort of oblivion. It appeared that even in the worst-case scenario, the safety risks associated to modified microorganisms were not worse than naturally occurring counterparts. On the contrary, the rule of thumb was that any man-made genetic modifications resulted in strains that were less fit to compete with the indigenous, native microbial population of wherever site the GEMs could go to [9]. This was good news for the assessment of risks but very bad for the incipient branch of Environmental Biotechnology based on deliberate release of GEMs for bioremediation, biomining or plant-growth promotion. These expectations, which peaked by 1989 [10], came to an end a few years later after realizing how difficult it was to alter the homeostasis of a pre-existing complex biosystem by just adding a new component — whether one/few genes or a new organism [9]. For a number of reasons, the issue of containment of GEMs has been somewhat silent for many years, in part because of the lack of accidents and in part because releasing modified bacteria has not been useful thus far as a sound bioremediation strategy [9]. This is changing with the onset of Synthetic Biology.
Section snippets
What new environmental risks are brought about by Synthetic Biology?
The early 2000s witnessed the birth of Synthetic Biology as conceptual and technical umbrella that adopts electric engineering, mechanical engineering and computation as the interpretative frame for biological objects [11, 12, 13, 14] This has allowed making amazing breakthroughs in our understanding of the relational logic of living objects as well as in the partial or complete redesign of such logic to create new-to-nature-properties. As a result, the concept genetic engineering stops being a
From synthetic auxotrophies to semantic containment
Engineering a GE agent of interest to become dependent on the external addition of a key metabolic intermediate or cell building block for growth and survival has been one of the most widespread stratagems to ensure containment within a desired spatiotemporal frame (called trophic containment). One of the outcomes of the 1975 Asilomar conference was in fact the proposition of using multi-amino acid auxotrophic Escherichia coli strains as the hosts of reference for recombinant constructs [1, 2].
Circuits and more circuits
Since the start of GE, the second grand strategy for containment of engineered microorganisms has involved genetic circuits that limit the survival of the agents at stake beyond specific physicochemical and/or nutritional niches. Typically, they involved a more or less complex network of transcriptional factors, cognate promoters, killer genes and antidotes, the balance of which in response to an environmental signal determined continued existence or programmed death [6]. Many of such earlier
Gene drives and molecular vigilantes
One of the most recent (and fascinating) technologies empowered by Synthetic biology is the so-called gene drive, which enforces inheritance of particular genes or gene variants through entire populations of species that undergo sexual reproduction (i.e. each individual acquires one set of chromosomes from each of the parents). The result is a sort of mutagenic chain reaction [60]. To this end, one individual of the target species is engineered to have the diploid version of one or more genomic
Outlook
While the conceptual rigour of the methodologies just discussed may appear to cover most of the containment needs, in reality there is still a considerable way ahead. As new (meta)genomes are sequenced, novel lethal genes and regulatory elements become available for designing more intricate conditional killing systems that could increase containment efficiencies. Circuits based on familiar biology will however never be absolute, as mutations will inevitably lead to the appearance of surviving
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
MS gratefully acknowledges the EC FP7 projects METACODE (289572) and ST-FLOW (289326). MS is indebted to the other members of the EC SCENIHR working group on synthetic biology for inspiring and challenging discussions. The work in VdL Laboratory is supported by Projects ARISYS (322797) and EMPOWERPUTIDA (635536) of the EU and ERANET Project CONTIBUGS.
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