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BUILT-IN BIOSAFETY DESIGN Ollie Wright - 29/04/13 INTRODUCTION • Two concerns from Asilomar: that outcompete indigenous organisms • GMMOs • Genetic contamination via horizontal gene transfer (HGT) 1975 Asilomar Courtesy of Google IGEM • iGEM 2012 • Physical containment • Amber suppression • Endonuclease bacteriocin toxins Weakness 1 & 2 X 3 5 4 BENCHMARKS 1x10-8 per cell generation Full thyA deletion Moe-Behrens, G. H. G., Davis, R., & Haynes, K. A. (2013). Preparing synthetic biology for the world. Frontiers Microbiol, 4, 5. doi:10.3389/fmicb.2013.00005 • Gain-of-function much harder to evolve than loss-of-function HOW TO IMPROVE? • Use microbes with low environmental retention times, rather than relying on kill switches • Remove conjugation/transduction machinery • Remove homologous sequences to minimise recombination • Transformation is harder to mitigate: • Minimise genes with an obvious selection advantage • Include genes with an obvious selection disadvantage FOCUS ON PLASMIDS? •3 advantages of plasmids over genomic integrants: • Easy control of gene dosage flanking homologous sequence as physically isolated from genome • No • Imperfect retention; reversion of host to near wild-type CONDITIONAL ORIGIN OF REPLICATION COR • Initiation of replication protein provided in trans to origin sequence • DIAL strains from Anderson lab • E.g. ColE2, R6K ca rg o • rep RBS COR auxo Kittleson, J. T., Cheung, S., & Anderson, J. C. (2011). Rapid optimization of gene dosage in E. coli using DIAL strains. J Biol Eng, 5, 10. doi:10.1186/1754-1611-5-10 Chromosome AUXOTROPHY auxo Host strain auxotrophic for essential metabolite (rich media compatible) • Metabolic gene provided in trans by plasmid • ca rg o • E.g. thyA (thymidine), dapA (diaminopimelic acid) or thiL (thiamine pyrophosphate) rep antitoxin auxo COR F R T selection F R T Chromosome TOXIN-ANTITOXIN auxo Toxin-antitoxin pair • Usually bicistronic, but can shift antitoxin to chromosome • E.g. Zeta/Epsilon (cell wall synthesis), Kid/Kis (endoribonuclease) antitoxin toxin rep ca rg o • COR F R T selection F R T Chromosome SYSTEM REDUNDANCY auxo COR • Auxotrophy • Toxin-antitoxin pair as bonus ca rg o • toxin COR rep antitoxin Chromosome ACCESS EFFICACY OF DESIGN • If selection coefficient is weak, traditionally need large number of microbial generations before sampling - in the order of 10 to 1,000 years, if ever... • As proof-of-principle, need to make a worst-case-scenario system with a strong selection coefficient - sample after hours/days • If a biosafety design passes this worst-case-scenario, a strong indicator of suitability THE FUTURE • Semantic containment: • Codon refactoring to shuffle natural genetic code GG G CCC Courtesy of Google • Xeno-nucleic acids that uses orthogonal machinery Schmidt, M., & de Lorenzo, V. (2012). Synthetic constructs in/for the environment: managing the interplay between natural and engineered biology. FEBS Lett, 586(15), 2199–2206. TAKE HOME MESSAGES • General recommendations for the here-and-now: • System redundancy - don’t rely on linear chains • Deletions preferable - regaining function is evolutionary more difficult than inactivation (i.e. kill switch) • Minimise fitness cost of incorporating biosafety to promote practical use ACKNOWLEDGEMENTS CSynBI, Imperial College http://tinyurl.com/synbiosafety http://www3.imperial.ac.uk/syntheticbiology Sponsors