Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies.

Comparison of genome-wide selection strategies to identify furfural tolerance genes in Escherichia coli.

Engineering both feedstock and product tolerance is important for transitioning towards next-generation biofuels derived from renewable sources. Tolerance to chemical inhibitors typically results in complex phenotypes, for which multiple genetic changes must often be made to confer tolerance. Here, we performed a genome-wide search for furfural-tolerant alleles using the TRackable Multiplex Recombineering (TRMR) method (Warner et al.

Genome-wide mapping of furfural tolerance genes in Escherichia coli.

Advances in genomics have improved the ability to map complex genotype-to-phenotype relationships, like those required for engineering chemical tolerance. Here, we have applied the multiSCale Analysis of Library Enrichments (SCALEs; Lynch et al. (2007) Nat.

Strategy for directing combinatorial genome engineering in Escherichia coli.

We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856-862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894-898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and pharmaceuticals, among other products.