(619b) Automated Design of Non-Repetitive Genetic Parts Using Non-Repetitive Parts Calculator and Its Application in Characterizing 4,350 Highly Non-Repetitive E.coli Promoters
AIChE Annual Meeting
Thursday, November 1, 2018 - 8:18am to 8:36am
To solve this challenge, we developed and experimentally validated the Non-Repetitive Parts (NRP) Calculator, an optimization algorithm that designs a large maximally non-repetitive toolbox of genetic parts according to a user-specified set of design constraints. The design constraints may be a degenerate nucleotide or amino acid sequence, a proscribed RNA structure, a set of context-dependent background sequences and/or a quantitative model of part function. The algorithm then uses advanced path-finding in k-mer sequence space to generate genetic parts that satisfy the design constraints while not sharing repeats above a threshold length L, dictated by the system construction and host specifications. From extensive benchmarking, we show that the NRP Calculator can generate over 100,000 non-repetitive (L = 14) genetic parts (each 100 bp long) in less than 7 minutes.
As a first experimental example, we applied the NRP Calculator to build extremely large toolboxes of non-repetitive Ï70E. coli promoter sequences (L = 10), offering tunable control over many gene expression levels in large genetic systems. The first toolbox contains 800 non-repetitive strong promoters, each with a consensus -35 and -10 hexamer. The second toolbox contains 3,500 non-repetitive promoters with varied strengths, containing -35 and -10 hexamers that deviate from the consensus sequence by 0 to 6 mismatches. The third toolbox contains 50 weak non-repetitive promoters with 12 mismatches in the hexamers. We measured the transcription rates of these 4,350 new non-repetitive promoters, utilizing chip-based oligo pool synthesis and a massively parallel reporter assay in E. coli NEB 5Î±, followed by DNA-Seq and RNA-Seq measurements. The entire library spanned a 2,591,738-fold transcription rate dynamic range and 2,858 promoters from the library had transcription rates higher than the standard J23100 promoter. Overall, the Non-Repetitive Parts Calculator promises to design and deliver a cornucopia of well-characterized genetic parts that can all be simultaneously combined to build extremely large genetic systems that will power the future of Synthetic Biology.