Comparison of FRUIT to pKD13-mediated recombineering.

a<p>Frequency with which candidate colonies were successfully verified.</p>b<p>Number of colonies/number of viable cells.</p>c<p>Relative efficiency of FRUIT as compared to pKD13.</p

Schematic of FRUIT method.

<p>(A) Schematic of FRUIT for introducing point mutations or deletions. PCR product is amplified from the recombineering template plasmid (pAMD001), incorporating flanking sequence with identity to the desired site of recombination. This PCR product is introduced into cells expressing λ recombinase proteins and recombinants are selected using the <i>thyA</i> marker (growth on media lacking thymine). A mutation can then be introduced by recombineering a second PCR product, selecting for recombinants using counter-selection of <i>thyA</i> (growth in the presence of trimethoprim). (B) Schematic of FRUIT for introducing FLAG tags. As above, except that loss of <i>thyA</i> occurs spontaneously due to homologous recombination of duplicate sets of FLAG tags.</p

FRUIT promoter swaps in MG1655 (<i>E. coli</i> K-12).

<p>(A) Schematic indicating the plasmid templates used for FRUIT. (B) Schematic indicating replacement of the <i>lacZYA</i> promoter with P_high, P_med, P_low or P_rha promoters. (C) β-galactosidase assay in Δ<i>lacZ</i> MG1655 and mutant strains with P_high, P_med or P_low driving expression of <i>lacZYA</i> (cells were grown without IPTG). (D) β-galactosidase assay in Δ<i>lacZ</i> MG1655 and a mutant strain with P_rha driving expression of <i>lacZYA</i>. Assays were performed ± rhamnose.</p

List of strains and plasmids.

<p>List of strains and plasmids.</p