TABLE 5.
Primers used in this studya
| Primer | Sequence (5′–3′) |
|---|---|
| Gene cloning | |
| EcAroG-Nde | CCAACCATATGAATTATCAGAACGACGATTTACG |
| EcAroG-D146N-RM | GGGGTGATCATATTGAGAAACTC |
| EcAroG-D146N-FM | GAGTTTCTCAATATGATCACCCC |
| EcAroG-R | CAAAGCTTTTACCCGCGACGCGCTTTTAC |
| AroG-RM-Nde | GCTATTATCCATGTGCGGATCG |
| AroG-FM-Nde | CGATCCGCACATGGATAATAGC |
| EcPheA-F | CCAACCATATGACATCGGAAAACCCGTTAC |
| EcPheA-R | CACTCGAGTCAGGTTGGATCAACAGGCAC |
| EcTyrA-F | CCAACCATATGGTTGCTGAATTGACCGC |
| EcTyrA-R | CACTCGAGTTACTGGCGATTGTCATTCGCC |
| EctktA-Nde | CCAACCATATGTCCTCACGTAAAGAGCTTGCC |
| EctktA-Xho | CACTCGAGTTACAGCAGTTCTTTTGCTTTC |
| ppsA-F | CCAACCATATGTCCAACAATGGCTCGTC |
| ppsA-R | CACTCGAGTTATTTCTTCAGTTCAGCCAGG |
| EcAroA-F | CAACACCATGGAATCCCTGACGTTACAAC |
| EcAroA-R | CAAAGCTTTCAGGCTGCCTGGCTAATCC |
| AroB-opt-gib-F | GTTTAACTTTAAGAAGGAGATATACATATGGAGCGTATTGTCGTTACTC |
| AroB-gib-R | GTGGTGGTGGTGCTCGAGTTACGCTGATTGACAATCGGC |
| AroC-gib-F | GTTTAACTTTAAGAAGGAGATATACATATGGCTGGAAACACAATTGGAC |
| AroC-gib-R | GTGGTGGTGGTGCTCGAGTTACCAGCGTGGAATATCAGTC |
| AroD-gib-F | GTTTAACTTTAAGAAGGAGATATACATATGAAAACCGTAACTGTAAAAGATC |
| AroD-gib-R | GTGGTGGTGGTGCTCGAGTTATGCCTGGTGTAAAATAGTTAATAC |
| AroE-gib-F | GTTTAACTTTAAGAAGGAGATATACATATGGAAACCTATGCTGTTTTTGG |
| AroE-gib-R | GTGGTGGTGGTGCTCGAGTCACGCGGACAATTCCTCC |
| AroL-gib-F | GTTTAACTTTAAGAAGGAGATATACATATGACACAACCTCTTTTTCTGATC |
| AroL-gib-R | GTGGTGGTGGTGCTCGAGTCAACAATTGATCGTCTGTGCC |
| ALCorEDB-F | GAAATTAATACGACTCACTATAGGGGAATTG |
| ALC-aroA-R | TCAGGCTGCCTGGCTAATC |
| ALC-aroL-F | GATTAGCCAGGCAGCCTGACTTTAAGAAGGAGATATACATATGACAC |
| ACL-aroL-R | GGATGGCCTCCTTTAGATCCTCAACAATTGATCGTCTGTGC |
| ALC-aroC-F | GGATCTAAAGGAGGCCATCCATGGCTGGAAACACAATTGG |
| ALCorEDB-R | AGCCAACTCAGCTTCCTTTC |
| aroALC-F1 | ATGGAATCCCTGACGTTACAAC |
| aroALC-R1 | TTACCAGCGTGGAATATCAGTCTTC |
| pET21a-ALC-F | CTGATATTCCACGCTGGTAACTCGAGCACCACCACCACC |
| pET21a-ALC-R | GTAACGTCAGGGATTCCATGGTAATATCTCCTTCTTAAAGTTAAAC |
| EDB-aroE-R | CATATGTATATCTCCTTCTTAAAGTCACGCGGACAATTCCTCC |
| EDB-aroD-F | CGTGACTTTAAGAAGGAGATATACATATGAAAACCGTAACTGTAAAAGACC |
| EDB-aroD-R | GGATGGCCTCCTTTAGATCCTTATGCCTGGTGTAAAATAGTTAATAC |
| ALC-aroB-F | ATGGAGCGTATTGTCGTTACTCTGGGCGAACGTAGCTACC |
| EDB-aroB-F2 | GGATCTAAAGGAGGCCATCCATGGAGCGTATTGTCGTTACTC |
| aroEDB-F1 | ATGGAAACCTATGCTGTTTTTGG |
| aroEDB-R1 | TTACGCTGATTGACAATCGGC |
| pET21a-EDB-F | CGATTGTCAATCAGCGTAACTCGAGCACCACCACCACC |
| pET21a-EDB-R | CAAAAACAGCATAGGTTTCCATATGTATATCTCCTTCTTAAAGTTAAAC |
| aroLC-DB-F | CTTAAAGTTAAACAAAATTATTTCTAGAGG |
| aroLC-R | AAGGAGATATACATATGACACAACCTC |
| aroAC-F | CTTAAAGTCAGGCTGCCTGGC |
| aroAC-R | AAGGAGGCCATCCATGGCTGGAAAC |
| aroAL-F | TCAACAATTGATCGTCTGTGCC |
| aroAL-ED-R | CTCGAGCACCACCACCAC |
| aroDB-R | AAGGAGATATACATATGAAAACCGTAAC |
| aroEB-F | TCACGCGGACAATTCCTCC |
| aroEB-R | GGATCTAAAGGAGGCCATCCATG |
| aroED-F | TTATGCCTGGTGTAAAATAGTTAATACC |
| Amplification of integration cassette | |
| Delta-adhE-F | CGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATTATGATTCCGGGGATCCGTCGACC |
| Delta-adhE-FRT-R | CCGTTTATGTTGCCAGACAGCGCTACTGATTAAGCGGATTTTTTCGCTTTATTCGCCAATCCGGATATAG |
| Delta-ldhA-F | TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGATTCCGGGGATCCGTCGACC |
| Delta-ldhA-FRT-R | CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGCAATTCGCCAATCCGGATATAG |
| Delta-pflBA-F | CGAAGTACGCAGTAAATAAAAAATCCACTTAAGAAGGTAGGTGTTACATGATTCCGGGGATCCGTCGACC |
| Delta-pflBA-FRT-R | CTCAATAAAGTTGCCGCTTTACGGGGAAATTAGAACATTACCTTATGACCATTCGCCAATCCGGATATAG |
| Delta-pflDC-F | CTTCTCCCGCTCGCAAGGGCGGGTTCGCTTTCCCACAGGAGTTCCTCATGATTCCGGGGATCCGTCGACC |
| Delta-pflDC-FRT-R | AGGCGGTTACTGCCACCAGGTATGCCATTTTAACCTCCCACGGTAACCTGATTCGCCAATCCGGATATAG |
| Delta-pykF-F | GAAAGCAAGTTTCTCCCATCCTTCTCAACTTAAAGACTAAGACTGTCATGATTCCGGGGATCCGTCGACC |
| Delta-pykF-FRT-R | GATATACAAATTAATTCACAAAAGCAATATTACAGGACGTGAACAGATGCATTCGCCAATCCGGATATAG |
| Delta-pykF-R | GATATACAAATTAATTCACAAAAGCAATATTACAGGACGTGAACAGATGCTGTAGGCTGGAGCTGCTTCG |
| Delta-ascF-F | GACTGATAACAACTACATCTACCCTACTGATAACAGGATAAAATCCGATGATTCCGGGGATCCGTCGACC |
| Delta-ascF-FRT-R | ACTTTCTGGAAATACTGACATTTTCATCCTCAATTAAGACTTACTTCTTTATTCGCCAATCCGGATATAG |
| Delta-ascF-R | ACTTTCTGGAAATACTGACATTTTCATCCTCAATTAAGACTTACTTCTTTTGTAGGCTGGAGCTGCTTCG |
| Delta-pykA-F | TTTCATGTTCAAGCAACACCTGGTTGTTTCAGTCAACGGAGTATTACATGATTCCGGGGATCCGTCGACC |
| Delta-pykA-FRT-R | TGGCGTTTTCGCCGCATCCGGCAACGTACTTACTCTACCGTTAAAATACGATTCGCCAATCCGGATATAG |
| Delta-pykA-R | TGGCGTTTTCGCCGCATCCGGCAACGTACTTACTCTACCGTTAAAATACGTGTAGGCTGGAGCTGCTTCG |
| Delta-tyrB-F | GTTTATTGTGTTTTAACCACCTGCCCGTAAACCTGGAGAACCATCGCGTGATTCCGGGGATCCGTCGACC |
| Delta-tyrB-R | GCTGGGTAGCTCCAGCCTGCTTTCCTGCATTACATCACCGCAGCAAACGCTGTAGGCTGGAGCTGCTTCG |
| Delta-mtlA-F | TCGGGCTTCCAGCCTGCGCGACAGCAAACATAAGAAGGGGTGTTTTTATGATTCCGGGGATCCGTCGACC |
| Delta-mtlA-FRT-R | CTTCTCCATGTGGAGAGGGTGGGATTGGATTACTTACGACCTGCCAGCAGATTCGCCAATCCGGATATAG |
| Delta-tyrR-F | GTGTCATATCATCATATTAATTGTTCTTTTTTCAGGTGAAGGTTCCCATGATTCCGGGGATCCGTCGACC |
| Delta-tyrR-FRT-R | TGGTGTTGCACCATCAGGCATATTCGCGCTTACTCTTCGTTCTTCTTCTGATTCGCCAATCCGGATATAG |
| Delta-ldhA-feaB-F | AAATATCTGTTTTAACTAATTGGCGTTGCAGTACATGCAACGCCAATTAGATTCCGGGGATCCGTCGACC |
| Delta-ldhA-feaB-FRT-R | TGCCGTTTTTTACTTATGAGCGAACCAGATTAATACCGTACACACACCGAATTCGCCAATCCGGATATAG |
| Check for integration | |
| k1 | CAGTCATAGCCGAATAGCCTC |
| k2 | CGGTGCCCTGAATGAACTGC |
| U-adhE | CTGAATCACGGTTAGCTCCGAAG |
| D-adhE | TGCAGGCCGTGCCAGTCATCC |
| U-ldhA | CTCCGTCTGCGGCAATTTC |
| D-ldhA | GTCTGTTTTGCGGTCGC |
| U-pflBA | CCGCAAATGGTCAATGGGGAC |
| D-pflBA | CGTTATTCTCCAGAGGTTCATCAGC |
| U-pflDC | CAGTGGTGACGGCTGTTTGTG |
| D-pflDC | CGTTATTGAAAGGCTATGACCTGAAG |
| U-pykF | CAAAATCAGACAAATAACGC |
| D-pykF | GAGCTGCGTCATCTTTAG |
| U-ascF | GTAATTCACTAAAATAAATGCCGTGG |
| D-ascF | CGTAGCGGCTGAAAAACTCCACC |
| U-pykA | ACGCATGAGTTGTATGAATTGTAG |
| D-pykA | GTACTGGGGATATTATTTACCCG |
| U-tyrB | CAGTGCTGGTGAACGGTCG |
| D-tyrB | CGCTTTGCTGTTTTGCCGAG |
| U-mtlA | GCCAGAAGGGAGTCAGGCTG |
| D-mtlA | GATCAACGACATCATCACCAATGC |
| U-tyrR | GTTTAATTAATCGCATCGCCACGC |
| D-tyrR | CGTAAGTTTAACCAACTGGCAACTG |
| U-ldhA-feaB | CAGCGTTAACTGGTTCGCGGTC |
| D-ldhA-feaB | GCGCCAACAGATTTTCTG |
a
Primers for amplification of integration cassette were selected by target chromosomal locus. For example, the primer pair delta-pykF-F/delta-pykF-FRT-R was selected when a gene would be integrated into the pykF locus of the chromosome (a gene cloned by pET21a-FRT was used as a template DNA). In such cases, gene integration was checked after Red recombination by the primer pair k1/U-pykF (about 1,000 bp) in addition to the primer pair k2/D-pykF (about 1,200 bp plus the length of a gene). For gene deletion, delta-pykF-R was used instead of delta-pykF-FRT-R, along with pKD13 as a template DNA.