Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice
Our procedure is identical to the Red-swap described in reference  with the exception that we re-engineered the original insert to include an in-frame T7 RNA polymerase promoter (PT7), and the sequences used for recombination are longer. A gene, identified in the schematic as genE, is targeted for deletion. Two 65mer primers (red) are used to amplify the region containing the antibiotic resistance cassette, the PT7, and the FRT sites (among other elements) from the plasmid pCLF4. The resulting PCR product has 45 base sequences at each end that are homologous to sequences near the 5′ and 3′ ends of the targeted ORF. Transformation of these PCR products into ATCC14028 expressing lambda Red recombinase in trans leads to a recombination event resulting in the swap-in of the PCR product, and swap-out of the targeted gene. A ribosomal binding site (RBS) and a downstream ATG start codon near the 3′ end of the inserted sequence ensures that a 12 amino acid peptide is made from any RNA that is transcribed in this strand to reduce polar effects. Targeted mutants in many genes were pooled and used for Array-based analysis of cistrons under selection (ABACUS). Finally, antibiotic resistance markers in targeted genes can be removed using the FLP recombinase resulting in a gene encoding a mini-protein of the first ten amino acids of GenE, 39 amino acids from the inserted DNA (called a “scar”), and the last nine amino acids of GenE, and retaining the PT7.