TABLE 1.
Oligonucleotides used in this study
| Gene target | Primer | Sequence |
|---|---|---|
| λ Red knockoutsa | ||
| purA | OKD499 | GTAACAACGTCGTCGTACTGGGCACCCAATGGGGTGACGAAGgtgtaggctggagctgcttc |
| OKD500 | TACGCGTCGAACGGGTCGCGCAGAATCATGGTTTCAGTACGAatgggaattagccatggtcc | |
| OKD521 | ggtaacaacgtcgtcgtactg | |
| OKD522 | tacgcgtcgaacgggtcgcgc | |
| rpoS | OKD536 | TTTTGCTTGAATGTTCCGTCAAGGGATCACGGGTAGGAGCCAgtgtaggctggagctgcttc |
| OKD537 | CAGCCTCGCTTGAGACTGGCCTTTCTGACAGATGCTTACTTAatgggaattagccatggtcc | |
| OKD544 | gttgtcggtagcagacggtct | |
| OKD545 | gaaccagttcaacacgcttgc | |
| ndk | OKD540 | ACGCTGGTACAGACAACAACAGAACAATTTACAGAGGTAAAAgtgtaggctggagctgcttc |
| OKD541 | CGGATGCCACGTTTGCACGCGGCATTTACGAAATTATTAACGatgggaattagccatggtcc | |
| OKD542 | gacatatgctattccggcctc | |
| OKD543 | caatagtcaacggccctgttg | |
| relA | OKD557 | ATTTGCCGATTTCGGCAGGTCTGGTCCCTAAAGGAGAGGACGgtgtaggctggagctgcttc |
| OKD558 | TCTACATTGTAGATACGAGCAAATTTCGGCCTAACTCCCGTGcatatgaatatcctccttag | |
| OKD553 | gcattaacgtagccgggatcc | |
| OKD554 | ctggatatgttcccacacacg | |
| spoT | OKD559 | GCTATTGCTGAAGGTCGTCGTTAATCACAAAGCGGGTCGCCCgtgtaggctggagctgcttc |
| OKD560 | CCTGGCGAGCATTTCGCAGATGCGTGCATAACGTGTTGGGTTcatatgaatatcctccttag | |
| OKD555 | caggaacagcaagagcaggaa | |
| OKD556 | cagatgcgtgcataacgtgtt | |
| Kmr | OKD293 | ccaagctcttcagcaatatcac |
| Complementationb | ||
| purD | OKD429 | atggatccactgactgctgcattccc |
| purH | OKD430 | gagtcgacgtcagttagggatcacg |
| purF | OKD433 | aacggccgctggcgcgtcttatcagg |
| OKD434 | aacggccgactttgcaggatttcgg |
The 62-base-long oligonucleotides used for λ Red recombineering were designed with two domains: the segment homologous to the gene targeted for knockout is capitalized, and the segment homologous to the Kmr gene of pKD4 is in lowercase (11). The oligonucleotides for relA and spoT knockouts differ because they include the FRT sites. Oligonucleotides OKD521-22, 542-5, and 553-6 were used for PCR confirmation of the λ Red knockout, and they anneal to sequences up- and downstream of the target gene. OKD293 anneals within the Kmr gene and was used as an internal primer, together with a flanking primer, to confirm replacements.
Oligonucleotides used for complementation contain EagI, BamHI or SalI restriction sites (boldface), to facilitate cloning of the amplified gene into pBR322, for complementation studies.