The advent of highly efficient genome engineering technologies that enable site-specific manipulation of chromosomes offers exciting opportunities for industrial strain engineering. However, the utility of rapidly constructed strain libraries is limited by the lack of downstream analysis tools that can effectively assess the combinatorial diversity and mapping of genotype-phenotype relationships at distal sites in the genome.
A University of Colorado Boulder research group has developed a method to track combinatorial mutants with single genotype resolution at the scale of up to several dozen sites. This approach, barcoded tracking combinatorial engineered libraries (bTRACE), works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This technology utilizes a systematic approach, an automated software based primer design tool, and an understanding of multiplexed linking PCR chemistry to rapidly and inexpensively genotype combination of mutations that occur in single genotypes or diverse populations.
Zeitoun RI, Pines G, Grau WC, Gill RT. Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies. ACS Synth Biol. 2017 Apr 21;6(4):619-627. Epub 2017 Jan 24. PMID: 28103008.
Zeitoun RI, Garst AD, Degen GD, Pines G, Mansell TJ, Glebes TY, Boyle NR, Gill RT. Multiplexed tracking of combinatorial genomic mutations in engineered cell populations. Nat Biotechnol. 2015 Jun;33(6):631-7. Epub 2015 Mar 23. PMID: 25798935.
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