Multilayered genetic safeguards limit growth of microorganisms to defined environments

Published online 7 January 2015 Nucleic Acids Research, 2015, Vol. 43, No. 3 1945–1954 doi: 10.1093/nar/gku1378 Multilayered genetic safeguards limit growth of microorganisms to defined environments Ryan R. Gallagher1,2,† , Jaymin R. Patel1,2,† , Alexander L. Interiano1 , Alexis J. Rovner1,2 and Farren J. Isaacs1,2,* 1 2 Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA and Systems Biology Institute, Yale University, West Haven, CT 06516, USA Received November 21, 2014; Revised December 20, 2014; Accepted December 20, 2014 ABSTRACT Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping ‘safeguards’––engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome––to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10−12 . The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species. INTRODUCTION Since the advent of genetic engineering (1), genetically modified organisms (GMOs) have enabled functional testing of mutations and production of valuable pharmaceutical or industrial compounds (2). Advances in synthetic biology have led to GMOs with increasingly complex functions including production of fuels and medicines (2), and genetic circuits that can sense and respond to changing environments (3). As sophisticated GMOs expand to applications in open systems such as environmental (4) or clinical settings (5), there is a growing need for intrinsic biocontainment strategies––robust genetic safeguards that conditionally restrict the host cell’s viability to defined environments (6). Specifically, an intrinsic biocontainment strategy able to restrict growth to environments containing synthetic small molecules could prevent a GMO’s dissemination and enhance its safety. Prior strategies for biocontainment are based on designs to control cell growth by engineered auxotrophy (7), essential gene regulation (8) or toxin expression (9,10). While the best-performing safeguards reach the 10−8 NIH standard (11) for escape frequency of recombinant microorganisms (12,13), each approach carries risk. Auxotrophy can be complemented by metabolite cross-feeding (14) or by environmental availability of essential small molecules, yielding strains that grow in rich media and natural environments. Leaked expression of essential genes can permit viability (8) and mutations lead to loss of toxins (15). Attempts to implement redundant safeguards reduce the risk of escape, but at the price of decreased fitness (16,17), leading to a growth advantage for escaping mutants. We propose that genetic safeguards possess three crucial properties: (i) low escape frequency, (ii) robustness and (iii) modularity. Safeguards with low escape frequency will prevent the rise of mutants escaping defined media and limit growth in the wild. Robust safeguards retain wild-type levels of fitness while also maintaining containment in diverse growth conditions. This crucial requirement demands that low escape frequency safeguards maintain their performance in rich or diverse environments, where provision of auxotrophic metabolites by other community members is possible. Modularity will allow many different strategies to be combined in one strain enabling multilayered safeguards, or for those safeguards to be transferred to different organisms enabling portability. To satisfy these three requirements, we present a strategy based on the staged introduction of independently acting safeguards that use auxotrophy, engineered riboregulation and engineered addiction to * To whom correspondence should be addressed. Tel: +1 203 737 3156; Fax: +1 203 737 3109; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1946 Nucleic Acids Research, 2015, Vol. 43, No. 3 MATERIALS AND METHODS Plasmids––cloning and DNA synthesis and assembly Basic molecular biology techniques were used in plasmid construction. Riboregulated essential gene plasmids (18) were constructed by amplifying genes from Escherichia coli using primers to add KpnI and HindIII restriction sites (Supplementary Table S1). Those fragments were cloned between KpnI and HindIII sites in the pZE21Y12a12C vector (Supplementary Figure S1). For all cloning, insert amplicons were purified using spin columns (QiaGen), digested with restriction endonucleases (NEB), agarose gel purified, extracted (QiaGen), ligated (Quick Ligase, NEB), then transformed by electroporation with parameters 1800 V; 25-␮F capacitance; 200- resistance; in 1-mm cuvettes (Bio-Rad). The pBAD21G plasmid was created by cloning the para BAD promoter amplified from pBAD-HisB (Invitrogen) between XhoI and KpnI in plasmid pZE21G (18). Toxin gene plasmids were created by cloning into KpnI- and HindIII-cut pBAD21G or by Gibson Assembly (NEB) into the same vector. For Gibson Assembly (20), the cloning vector was linearized by amplification using primers annealing near KpnI and HindIII sites. Toxin inserts were amplified using primers that added homologies to the vector termini; these homology arms were designed to anneal to the vector with a Tm = 60◦ C (∼25 bp). Toxin genes were either amplified from the E. coli chromosome or were synthesized in codon-optimized form (gBlocks, IDT) (Supplementary Table S2). Supplemental repressor plasmids were made by Gibson Assembly (20) into the pBAD21G vector using lacI or tetR genes amplified from E. coli and using synonymously recoded fragments obtained from IDT. Polymerase chain reaction (PCR) reactions were carried out using HotStart HiFi Mastermix enzyme (Kapa Biosystems) on a C1000 thermal cycler (Bio-Rad). The following amplification protocol was used: 3 min at 95◦ C initial denaturation; 30 cycles of 20 s at 98◦ C, (...truncated)