TABLE 2.
Oligonucleotides used in the study
| Function and name of oligonucleotide | Sequence (5′ to 3′) |
|---|---|
| Primers for PCR amplification of EMSA probe with biotin labeling | |
| EMSA3880-3884F | AGCCAGTGGCGATAAGTCGTCGCTGCACACCCGCATGTC |
| EMSA3880-3884R | AGCCAGTGGCGATAAGTGGTGTCCGGGCCGATCACTGAG |
| Primers for real-time RT-PCR | |
| RT3880F | CGGTCAACAAGGTCGGCTTCAC |
| RT3880R | ATCAGGTTGCCCAGCGAGGT |
| RT3882F | GGGTGCTGGTCATCGACAACAC |
| RT3882R | TCGTCCTCGGTCATGCCCATGT |
| RT3884F | CTCGGCTACTACAGCATCGGTTC |
| RT3884R | ATCGTGGAGGTCTGCGGGAT |
| RT8101F | GTTGCGATGCCGTGAGGT |
| RT8101R | CGGGTGTTACCGACTTTCA |
| Primers for identification of cotranscription of SACE_3880–3884 | |
| sqRT3880/3881F | TCAGCGTCAACAACTCCACCTC |
| sqRT3880/3881R | AGGCAGCCGGTAGTCCATCT |
| sqRT3881/3882F | AGATCACGCCCGACCACAAC |
| sqRT3881/3882R | GGAGACGCACACGACCTTCT |
| sqRT3882/3883F | CCGACTTCATCGCCAACGCA |
| sqRT3882/3883R | TCAGGTGGATCGGGTTGAGCAT |
| sqRT3883/3884F | GCACCGCAAGCAGAACACCAA |
| sqRT3883/3884R | CGGACGAACCGATGCTGTAGTAG |
| Guide sequence for CRISPR/Cas9-mediated deletion of gdhA1 and BcdhE1 gene | |
| 3882sgRNA | CGTGCCCGAGTCGCTCGGCGGGG |
| 3884sgRNA | GCGAAAACCCTGGTGCGCCACGG |
| Primers of homologous arm for CRISPR/Cas9-mediated deletion of gdhA1 and BcdhE1 gene | |
| 3882upHaF | ATTACCCTGTTATCCCTAAAGCTTCGGTTTCGAGTCCGTGCTCGGCACGATCGC |
| 3882upHaR | GTGTTGTCCCGGGACCGCACACGACCTTCTCCGGGCCCCACTCGT |
| 3882dwHaF | GGAGAAGGTCGTGTGCGGTCCCGGGACAACACCGCCGTCGTGCTC |
| 3882dwHaR | AACGACGGCCAGTGCCAAGCTTCTTGTGCCGTCCGGCCGAGACCGGCTC |
| 3884upHaF | ATTACCCTGTTATCCCTAAAGCTTGGACGGTCTCGACGTCGGGTACTGGCTGG |
| 3884upHaR | ATGGTCTCCTCGTCCAGACTCGTGCCCGGACGAACCGATGCTGTAGTAGC |
| 3884dwHaF | CGTCCGGGCACGAGTCTGGACGAGGAGACCATCGAGCAGGAGGCGC |
| 3884dwHaR | AACGACGGCCAGTGCCAAGCTTACGCGTCGACGCAGGTCAGTTCGGCCGACA |
| Primers for identification of gdhA1-deleted and BcdhE1 gene-deleted mutants | |
| Qc3882JLF | GCACGGTCAACAAGGTCGGCTTC |
| Qc3882JLR | GAAGCTGCACACCACGACCGAGT |
| Qc3884JLF | ATGGGCATGACCGAGGACGACG |
| Qc3884JLR | GACGAAGGCGAGCGTGAAGTGCTT |
| Primers for construction of the complementary strain | |
| 3396compF | CCAATGCATCTCCGACGGGGCGACCGAGGAGTAG |
| 3396compR | GCTCTAGACTACCCATAAGTGAATGCCCGATAGGCATCGGTGC |
| P3880-3884F | CCAATGCATGATGCGCAGCACGGTGCCACACGG |
| P3880-3884R | GGAATTCCATATGCGACATTCCACCTCTTCCTCGTCGCTGATC |
| 3882compF | GGAATTCCATATGCAGGAGATCGACGAGTGGGGCCCGGAGAAG |
| 3882compR | GCTCTAGATTACGCGGTCACGGCCTCGGG |
| 3884compF | GGAATTCCATATGAACGGCCGCGAGCCCATCGACG |
| 3884compR | GCTCTAGATCAGTGCTTGACCAGGCGCAGCG |
| Primers for construction of the overexpression strain | |
| 3880-3884overF | GGAATTCCATATGGAGTCGACGTTGAGCTCAGTGATCGGC |
| 3880-3884overR | GCTCTAGATCAGTGCTTGACCAGGCGCAGCG |
| Primers for construction of the E3ΔpccD strain | |
| pUC3396upF | CCCAAGCTTATGACCGCGAACTCCGAGACGCTCGAC |
| pUC3396upR | GCTCTAGAGTGCCGGTAGAGCGCGGAGGGC |
| pUC3396dwF | CGGGGTACCGGTTCATCGACATCCTGGTCACCGAGGTC |
| pUC3396dwR | CCGGAATTCGGCGGATGATGCCGTTCCACTCCTGC |
| Primers for E3ΔpccD strain confirmation by PCR | |
| UPF | GTACGCGGTTGAGGTGACCAGGAACTGCGG |
| Ut1 | CAGAACATACCGGTCCGCCTCATCGACTCCTCG |
| Dt1 | CGGAGAGAACGACGGGAAGGGAGAAGACGTAACC |
| DWR | CACGCCAGGTTGATGTCGGCACCGAGG |
| fd terminator sequence | CCCGGGAACCCGGCCGCGTCCGGCGCCCCCGCCGCCTTCGACGAGATCCCCGCAAAAGCGGCCTTTGACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCTGGGGAATTC |
| Primers for construction of the PermE-egfp-fd cassette | |
| egfp-ermEF | AGCTTTGATATCGCGAGTGTCCGTTCGAGTGGCGG |
| egfp-ermER | CTCGCCCTTGCTCACCATCGCTGGATCCTACCAACCGGC |
| egfp-egfpF | GACAATCGTGCCGGTTATGGTGAGCAAGGGCGAGGAGCT |
| egfp-egfpR | CGGCCGGGTTCCCGGGTTACTTGTACAGCTCGTCCATGCCGAG |
| egfp-fdF | CGAGCTGTACAAGTAACCCGGGAACCCGGCCGC |
| egfp-fdR | CCGGAATTCCCCAGATCTAAAGTTTTGTCGTCTTTCCAG |
| Primers for construction of the PermE-Sencas9-fd cassette | |
| cas9-ermEF | AGCTTTGATATCGCGAGTGTCCGTTCGAGTGGCG |
| cas9-ermER | TGTACTTCTTGTCCATCGCTGGATCCTACCAACCGGC |
| cas9-cas9F | TTGGTAGGATCCAGCGATGGACAAGAAGTACAGCATCGGCCTG |
| cas9-cas9R | CGGCCGGGTTCCCGGGTCAGTCGCCGCCGAGCTGG |
| cas9-fdF | GCTCGGCGGCGACTGACCCGGGAACCCGGCCGC |
| cas9-fdR | CCGGAATTCCCCAGATCTAAAGTTTTGTCGTCTTTCCAG |
| Cassette sequences | |
| PermE-3882sgRNA | GCTCTAGAGCGAGTGTCCGTTCGAGTGGCGGCTTGCGCCCGATGCTAGTCGCGGTTGATCGGCGATCGCAGGTGCACGCGGTCGATCTTGACGGCTGGCGAGAGGTGCGGGGAGGATCTGACCGACGCGGTCCACACGTGGCACCGCGATGCTGTTGTGGGCTGGACAATCGTGCCGGTTCGTGCCCGAGTCGCTCGGCGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTAAGCTTGGG |
| PermE-3884sgRNA | GCTCTAGAGCGAGTGTCCGTTCGAGTGGCGGCTTGCGCCCGATGCTAGTCGCGGTTGATCGGCGATCGCAGGTGCACGCGGTCGATCTTGACGGCTGGCGAGAGGTGCGGGGAGGATCTGACCGACGCGGTCCACACGTGGCACCGCGATGCTGTTGTGGGCTGGACAATCGTGCCGGTTGCGAAAACCCTGGTGCGCCACGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTAAGCTTGGG |