21Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the 22 engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have 23 enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed 24 mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date 25 been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of 26 inducible transcriptional regulators in these strains also prevents concurrent control of genome 27 engineering tools and engineered functions. Here, we introduce a new recombineering platform strain,
28BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity 29 and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination 30 efficiency, and (3) four independent inducible regulators to control engineered functions. These 31 modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold 32 increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To 33 facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci,34 termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is 35 a platform to develop and optimize engineered cellular functions and can serve as a model to implement 36 comparable recombination and regulatory systems in other bacteria.
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INTRODUCTION
38The design-build-test (DBT) cycle is a common paradigm used in engineering disciplines. Within the 39 context of synthetic biology it is employed to engineer user-defined cellular functions for applications 40 such as metabolic engineering, biosensing, and therapeutics (1, 2). The rapid prototyping of engineered 41 functions has been facilitated by advances in in vitro DNA assembly, and plasmids have traditionally 42 been used to implement designs in vivo given their ease-of-assembly and portability. However, for 43 deployment in contexts beyond the laboratory such as large-scale industrial bioprocesses or among 44 complex microbial communities, plasmid-based circuits suffer from multiple limitations: high intercellular 45 variation in gene expression, genetic instability from random partitioning of plasmids during cell division, 46 and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or 47 is economically infeasible (3, 4). These shortcomings can be ameliorated once a design is transferred 48 from a plasmid to the host genome, which offers improved genetic stability and lower expression 49 variation (5) along with reduced metabolic load (6). However, behaviors optimized for plasmid contexts 50 often do not map predictably to the genome. As such, building and testing designs directly on the 51 genome can reduce the DBT cycle time and facilitate engineering cellular programs for complex 52 e...