TABLE 2.
Primers used for PCR amplificationa
| Primer group and gene name | Primer pair sequences (5′-3′) |
|---|---|
| Primers for fhuR gene deletion | |
| fhuR up | CCTGATTTGATTATTACAGTTGATAAAG, TGGCGACGAGGATCCGTAATTGTTTAAT |
| fhuR down | GAAGTCTACTTTGGATCCTATGAATTTTA, GGACTGATTGCTACTAGTATTCTTGTTCC |
| Primers for fhuR gene amplification | |
| fhuR | TAAGGACTTTATCATATGAATATTAAACAATTACG, GAGTATTTATTTTAGCTCTTCCGCAAAATTCATAG |
| Primers for promoter amplification | |
| cysD | CTTCGGGTCATCTGGATTGA, AATGTTGCTCGTAAACATCC |
| cysE | GGTAACTCCATGATAAAGCT, CAATCAGGACAATGAAGAAT |
| cysM | CCGCAGCGTTAATAATTTCTT, CATTCCACCACTAGCGGGTG |
| fhuR | GCTTAGGAGAATTTGATGCAG, CGCCTTCAATAGTTAAAGTCGC |
| metB2 | GGTGGTTCAGGAGGAGCATTAGC, GAACCGCATGAATTCCAGCAAG |
| plpA | GCAGCGATAGCAACAATTAC, CCGTTAATAAACGGACATTG |
| purH | ACCAAGTGGTCGTAAAGTTGA, ACGGCTTTCGTTCCACCCGTTG |
| yhcE | CAGCATTTGAACCTGAACGAGT, CTAAGTGCCACCACGAACGATT |
| yjgC | GAAGTTCAGTCACGAGCTGACA, CCGCACGAGCAACCTCAATATC |
| yriD | GCGATTAAGGCAATTGCAAGTG, AATGACTGGACTTGTCAACTCA |
| Primers for gene inactivation | |
| cysK | GATTCGGCAAGCCGAAGCAG, GCGGTCTCTAAAGCCTCATC |
| metB2 | GACTTGCTGGAATTCATGCGG, TCATCCCTCCAAAGGCAGAC |
| yhcE | CGTTCGTGGTGGCACTTAGA, CCTAACACAAGCAAGACATC |
| Primers for Q-PCRb | |
| cadA | AGATAAGGTGGTCGATTTACCG, CGTATCAAGTACCACTGCCTCA |
| cysD | CAGCGACTCAAGCGATGTACTA, TACTCCCAAGTGTTCCCAAGTG |
| guaC | TATGGGAGCAACAATGGTAATG, TTTGATATTCTGAAGCCGAACC |
| mesJ | AATGCACGTGATTTCCGTTATC, CCCTGAAGACTTCTCAAAGGAC |
| metA | CCTTGATAAACCGCATAATTCA, GAAGGACTGAAACCCAATCTTC |
| metB1 | GTTGCTAGAGCAAATTGTCCTG, CAACGACCTATCAACATCCAGA |
| metB2 | GCCTTGGAAGAATTAATTGCAG, TTCCACCATAAACATCATCAGC |
| tuf | CGCGAACGTGGTATCACA, GTCCATTTGGGCAGCACC |
| ydcD | TAGTGAACCGGCAAATAAGGTC, GAGCCAGATTTGGTTCTGGTAG |
| yjiB | ACATTGAAGCCGGAAATACTTG, AGTGGCCTCGATAATTTGACTG |
| ykhH | GGAGCAATCAGAAACCAAGAAC, CAATACCCACAAGAACAAGCAG |
| ymcF | ATGCTCAAGGTTCTGTTTCGAG, TTCGCTAAGTCAACGGTTGTAG |
| yrfD | GGTTGCTGCTATTGGTAACTCG, AATCATACAGTCCACCCTCACC |
| yrjB | TTTAGGCCTTGAAGATCTTGGT, TTCAAGCCCTTTGACTTTATCC |
| ytjE | CAATTCCTGGAACAACTCCTTC, CGCAGACTATGGAGTTTATGGA |
| Primers for start transcription mapping | |
| cysD | CAATGGCATCATAATCAATC, CCCGGATTGCCCAAAGTTTC |
| cysM | GCATCAATAATCTCAGCTCC, CATGAATTTCAGGATTTGCG |
| fhuR | CTAATTCAAGTTTATTGAGC, GAGCGATTTTGTTTATTGAG |
| metA | ACCATAGCGAGCATAAAGTG, CAACAAATATGCAGACTTGA |
| metB2 | CAGTGTTAAGGCATCATGAGC, GCTGAAATTTCCTTAATGTCC |
| plpA | GACGAATTGGTGAAATTGTTG, CCAATTGCTTTCAAATCACC |
| yhcE | GCCAACGGTCTGAACGACCATC, GGAATTAACGAAGGTGAAGG |
| yjgC | GTTCCAATCTTGAATTGAC, GCTGGATTACCAATTGTCG |
| Primers for gel retardation probe | |
| cysD | GATAGAGCGGAACGGCGCGT, AATGTTGCTCGTAAACATCC |
| cysM | GTTGAAGTTTAACAATTGGAG, CCGCAGCGTTAATAATTTCTT |
| fhuR | GCTTAGGAGAATTTGATGCAG, CGCCTTCAATAGTTAAAGTCGC |
| metA | CGCGGCATGAGATTGACCACC, GATTAGCCTGGATTCCCCTAG |
| metB2 | CACGTCGTGATGAAGCGGCT, CGGAACAGATACTGCTCCTG |
| plpA | CCTACGCTGGCATTACCCAG, CGAGAACCATTCCGTCAAGA |
| purH (int.) | CGCATTCCTTGTTTAAGGTCG, GTCACATGAAATCAATGACAG |
| yhcE | CCTTTGTATCAACTGGATTG, GTTGCCCTTCTGAATAAGCC |
| yjgC | CAAGTCGGACTTGACCAAGC, GAACCTGATGAGCATGCAAC |
| Primers for substitution analysis of metB2 promoter FhuR box | |
| metB2 (A) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (B) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTCTAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (C) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTACAAGATTTTTTTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (D) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTCCCCATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (E) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGACCCTTTTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| Primers for fhuR gene deletion | |
| metB2 (F) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATCCACTTTTTTTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (G) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTTTTTATGTATACAAAAACG |
| metB2 (H) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTTTTTATGTATCAAAAAACG |
| metB2 (I) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTTTTTATACCCAAAAAAACG |
| metB2 (J) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTTTTTCTGTATAAAAAAACG |
| metB2 (K) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTAACTTTCCCCATGTATAAAAAAACG |
| metB2 (L) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAATTCCACTTTTTTATGTATAAAAAAACG |
| metB2 (M) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGAACTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (N) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCAGACTTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (O) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGCCACATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (P) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAGAAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (Q) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATAACAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (R) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTATCGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (S) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTTACAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (T) | CACGTCGTGATGAAGCGGCT,TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTTCATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (U) | CACGTCGTGATGAAGCGGCT,TAAAGATATCACGAAAGTGTGCCCAGTGCTATAAGATTTTTCTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| metB2 (V) | CACGTCGTGATGAAGCGGCT, TAAAGATATCACGAAAGTGTGCCCAGTGCTCTAAGATTTTTTTATAGCAGAATTAACTTTTTTTATGTATAAAAAAACG |
| Primers for substitution analysis of cysD promoter FhuR box | |
| cysD (A) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCATTAAAAAATCATATCGATAG |
| cysD (B) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCAGTAAAAAATCATATCGATAG |
| cysD (C) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCATTAAAAAATCATGTCGATAG |
| cysD (D) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCATGGGGAAATCATATCGATAG |
| cysD (E) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCATTAAAGGGTCATATCGATAG |
| cysD (F) | CATGGACTTGCAAAGTGTCG, CAACCTTGAGTTGTATTTTTTTATTTCTACTTTTAAAAGTTCTATCAGCTCCATTAAAAAAGTGGATCGATAG |
a
The BamHI restriction sites designed for fhuR gene deletion in L. lactis IL1403 strain are in bold. The NdeI-SapI restriction sites designed for fhuR gene amplification used to purify the protein are in bold. int., internal fragment of the purH gene. The substitution bases designed into the FhuR box of the metB2 and cysD genes are underlined. (A), probe control without substitution; (B-V), probes with substitutions in the FhuR box.
b
Q-PCR, quantitative PCR.