Table 1.
Plasmids and oligonucleotides used in this study.
| Plasmid | Properties | Reference |
|---|---|---|
| pEKEx2 | KanR; expression vector with lacIq, Ptac and pUC18 multiple cloning site | Eikmanns et al. (1991) |
| pEKEx2-eyfp | KanR, pEKEx2 containing eyfp with artificial RBS, under control of Ptac | This study |
| pEKEx2-eyfp-asv | KanR, pEKEx2 containing eyfp, with artificial RBS, under control of Ptac, modified to include the gene sequence for the C-terminal C. glutamicum SsrA tag variation AAEKSQRDYAASV | This study |
| pEKEx2-eyfp-aav | Similar to pEKEx2-eyfp-ASV using the alternative tag AAEKSQRDYAAAV | This study |
| pEKEx2-eyfp-lva | Similar to pEKEx2-eyfp-ASV using the alternative tag AAEKSQRDYALVA | This study |
| pEKEx2-eyfp-laa | Similar to pEKEx2-eyfp-ASV using the native C. glutamicum tag AAEKSQRDYALAA | This study |
| pEKEx2-eyfp-Ec_laa | Similar to pEKEx2-eyfp-ASV using the native E. coli tag AANDENYALAA | This study |
| pJC1 | KanR, AmpR; C. glutamicum shuttle vector | Cremer et al. (1991) |
| pJC1-PgntK-eyfp | KanR, pJC1 containing eyfp under control of the promoter of gntK (153 bp) | This study |
| pJC1-PgntK-eyfp-asv | Similar to pJC1-PgntK-eyfp using eyfp modified to include the gene sequence for the C-terminal C. glutamicum SsrA tag AAEKSQRDYAASV | This study |
| pEKEx2-eyfp-tetR | KanR, pEKEx2 derivative containing yfp-tetR, encoding a eYFP–TetR fusion protein under the control of the Ptac | Frunzke et al. (2008a) |
| pEKEx2-gfp | KanR pEKEx2 containing gfpuv with artificial RBS, under control of Ptac | This study |
| pEKEx2-gfp-asv | KanR, pEKEx2 containing gfpuv, with artificial RBS, under control of Ptac, modified to include the gene sequence for the C-terminal C. glutamicum SsrA tag variation AAEKSQRDYAASV | This study |
| pEKEx2-gfp-aav | Similar to pEKEx2-gfpuv-ASV using the alternative tag AAEKSQRDYAAAV | This study |
| pEPR1-gfp | gfpuv, KanR, rep, per, T1 (T-trpE) (T-trpA), T2 (T-rrnB) (T-leuB); promoter probe vector | Knoppova et al. (2007) |
| Oligonucleotide | Sequence 5′ -> 3′a | Applicationb |
| eYFP-ASV-C.g.-EcoRI-rv | CGCGAATTCTTAAACTGATGCAGCGTAATCACGTTGGCTCTTTTCTGCTGCTCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp-Cg-ASV |
| eYFP-AAV-C.g.-EcoRI-rv | CGCGAATTCTTAAACAGCTGCTGCGTAATCACGTTGGCTCTTTTCTGCTGCTCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp-Cg-AAV |
| eYFP-LVA-C.g.-EcoRI-rv | CGCGAATTCTTAAGCTACTAAAGCGTAATCACGTTGGCTCTTTTCTGCTGCTCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp-Cg-LVA |
| eYFP-LAA-C.g.-EcoRI-rv | CGCGAATTCTTAAGCTGCTAAAGCGTAATCACGTTGGCTCTTTTCTGCTGCTCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp-Cg-LAA |
| eYFP-LAA-E.c.-EcoRI-rv | CGCGAATTCTTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCTGCTCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp-Ec-LAA |
| eYFP-EcoRI-rv | CGCGAATTCTTATCTAGACTTGTACAGCTCGTC (EcoRI) | Rv for eyfp |
| eYFP-RBS-BamHI-fw | CGCGGATCCAAGGAGATATGATATGGTGAGCAAGGGCGAGGAG (BamHI) | Fw for eyfp and destabilized variants |
| eYFP-ASV-C.g.-SalI-rv | CGCGTCGACTTAAACTGATGCAGCGTAATCACG (SalI) | Rv for eyfp-Cg-ASV |
| eYFP-AAV-C.g.-SalI-rv | CGCGTCGACTTAAACAGCTGCTGCGTAATCACG (SalI) | Rv for eyfp-Cg-AAV |
| eYFP-NdeI-fw | CGCCATATGGTGAGCAAGGGCGAGGAG (NdeI) | Fw for eyfp and destabilized variants |
| PgntK-BamHI-fw | CGCGGATCCACATACAGTCCCCGTGATGTGAC (BamHI) | Fw for promoter region of gntK |
| PgntK-NdeI-rv | CGC CATATGGTCTTATCCTTTCTTTGGTGGCG (NdeI) | Rv for promotor region of gntK |
| GFPuv-AAV-C.g.-EcoRI-rv | GCGCGATATCGAATTCTCATTAAACAGCTGCTGCGTAATCACGTTGGCTCTTTTCTGCTGCTTTGTAGAGCTCATCCATGCCATG (EcoRI) | Rv for gfpuv-Cg-AAV |
| GFPuv-ASV-C.g.-EcoRI-rv | GCGCGATATCGAATTCTCATTAAACTGATGCAGCGTAATCACGTTGGCTCTTTTCTGCTGCTTTGTAGAGCTCATCCATGCCATG (EcoRI) | Rv for gfpuv-Cg-ASV |
| GFPuv-Acc65I-fw | GCGCGGTACCGGTAGAAAAAATGAG (Acc65I) | Fw for gfpuv and destabilized variants |
| GFPuv-EcoRI-rv | GCGCGCGAATTCGAGAGTTATCTCGGCCAGCCAC (EcoRI) | Rv for gfpuv |
a
Some oligonucleotides were designed with restriction sites (underlined), ribosome binding sites (bold) and include SsrA-tag sequences (italic) as indicated.
b
Rv, reverse primer; Fw, forward primer for amplification.