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Overview of strategy. (A) The TRMR library (middle circle) is generated by introducing mutations into WT E. coli (inner circle). A selection is performed yielding data on high-fitness mutants (outer circle). (B) A few mutants are chosen to be targets for further study. (C) A recursive multiplex recombineering library is constructed, generating a large diversity of clones with one or more mutations. (D) A selection is performed on the library to yield the most tolerant clones.
