As the field of synthetic biology develops, real-world applications are moving from the realms of ideas and laboratory-confined research towards implementation. A pressing concern, particularly with microbial systems, is that self-replicating re-engineered cells may produce undesired consequences if they escape or overwhelm their intended environment. To address this biosafety issue, multiple mechanisms for constraining microbial replication and horizontal gene transfer have been proposed. These include the use of host-construct dependencies such as toxin-antitoxin pairs, conditional plasmid replication or the requirement for a specific metabolite to be present for cellular function. While refactoring of the existing genetic code or tailoring of orthogonal systems, e.g. xeno nucleic acids, offers future promise of more stringent 'firewalls' between natural and synthetic cells, here we focus on what can be achieved using existing technology. The state-ofthe-art in designing for biosafety is summarized and general recommendations are made (e.g. short environmental retention times) for current synthetic biology projects to better isolate themselves against potentially negative impacts. IntroductionSynthetic biology aims to design, model and apply modular whole-cell systems to provide solutions to various challenges (Khalil & Collins, 2010). Real-world applications of synthetic biology range from molecular biosynthesis in enclosed bioreactors (Martin et al., 2003) through to sensing and acting upon external cues during environmental release, such as for biosensors (French et al., 2011), bioremediation (Singh et al., 2011) and biomining (Brune &Bayer, 2012). The majority of research and development in synthetic biology has utilized microbes as the host cells, which, in comparison with multicellular organisms, are more rapid to engineer and easier to understand. As synthetic biology advances, however, concerns are being raised about adverse effects that synthetic microbes may have if more broadly used or released into the environment (Dana et al., 2012;Moe-Behrens et al., 2013). Could genetically modified microbes (GMMs) outcompete native species and disrupt habitats? Could altered or synthetic genetic material escape its host and contaminate indigenous organisms?These concerns echo old questions raised previously by the introduction of recombinant DNA technology (Berg & Singer, 1995). At the 1975 Asilomar conference, scientists agreed on a cautious approach, incorporating both physical and biological containment into experimental design to minimize environmental risks that cisgenics or transgenics may pose (i.e. sequences native to the host, or to another species, respectively) (Berg et al., 1975). Four decades later, these principles have so far ensured no significant disaster (Berg & Singer, 1995;Benner & Sismour, 2005). Following the recent demonstration of a working synthetic genome (Gibson et al., 2010), a high-profile review has reaffirmed that the same caution applies to the use of 'syngenic' material, i.e. no...
Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.
High-throughput screens that dispense with the need for expensive synthetic Aβ peptide would be invaluable for identifying novel anti-aggregants as potential treatments for Alzheimer's disease. A biosynthetic in vivo approach, using a recombinant fluorescent green fluorescent protein (GFP) reporter for the aggregation state of Aβ in Escherichia coli, has been reported by other workers. Here, inducible Aβ-GFP expression in E. coli was coupled to the concurrent constitutive production of a quasi-random peptide library to screen for anti-aggregant activity. To attempt to introduce greater robustness, mCherry was also co-expressed as an internal fluorescence standard to allow ratiometric comparison between samples. However, fluctuations in mCherry expression levels, as well as a low dynamic range of GFP output between positive and negative anti-aggregant peptides, highlighted limitations with the approach. Despite this, two novel peptides were identified that showed an equivalent in vitro anti-aggregant activity to that of epigallocatechin-3-gallate. Thus, although biosynthetic in vivo strategies show promise as screens for novel activities, unforeseen problems can arise because of the variability inherent in any biological system.
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