TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Characteristics | Reference |
|---|---|---|
| Strains | ||
| Escherichia coli EC1000 | RepA+ MC1000, Kmr; host for pORI28-based plasmids | 26 |
| Lactobacillus acidophilus NCFM | Human intestinal isolate | 6 |
| Lactobacillus acidophilus NCK1392 | NCFM containing pTRK669 | 32 |
| Lactobacillus acidophilus NCK1728 | NCFM containing deleted version of ORF LBA0395 (frc) | This study |
| Plasmids | ||
| pFrc | 1.42 kb containing ORF_LBA395 cloned into the BglII/XbaI sites of pORI28 | This study |
| pTRK837 | pFrc containing a 72-bp deletion introduced by inverted PCR and self-ligation | This study |
| Primers for gene replacementa | ||
| LFoX | GATCTCTAGA-ATGCGTTATAATTGG | This study |
| RFoB | GATCAGATCT-ATCGGCAACTTAAA | This study |
| LFoE | GATCGAATTC-TTGCCGTATTAAGT | This study |
| RFoE | GATCGAATTC-CACGTTGCATTAAA | This study |
| Primers for RT-QPCR | ||
| RTLact16SF | GTAGGGAATCTTCCACAATG | 8 |
| RTLact16R | TAGTTAGCCGTGACTTTCTG | 8 |
| Q-394-F | ATGACGGTTGCCGATACGAT | This study |
| Q-394-R | GCGCAATTATGACCGCCTTA | This study |
| Q-395-F | AAGGTCTGGAGCACGCTTAT | This study |
| Q-395-R | CCAGTTGGTCCTGTCCTTGA | This study |
| Q-396-F | TGATGCAGCAGATGCTGGAG | This study |
| Q-396-R | CAACCGTGCCGTGGAATTAT | This study |
| Q-397-F | TTGAGCCGCTCTTAGGCTGCAA | This study |
| Q-397-R | TAGCGAACGTCCTTCAGGGAAA | This study |
| Q-BSH-F | GTGAAGAGAGGAGGCTTGCATTT | This study |
| Q-BSH-R | AGGTATGGCCGGACTCAACTATC | This study |
| Q-BSH2-F | ACGCCGACGTTACTCCACATA | This study |
| Q-BSH2-R | GTGGAGTGTGTGCCAAGACAA | This study |
a
Dashes indicate introduction of restriction enzyme sites.