Table 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic | Source or reference |
|---|---|---|
| Strains | ||
| E. coli strains | ||
| BL21(DE3) | F−ompT gal dcm lon hsdSB(rB− mB−) λ(DE3) | 27 |
| DH1 | endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK− mK+) λ− | 19 |
| LT-ΔfadE | DH1 ΔfadE with pKS1 | 26 |
| EGS084 | LT-ΔfadE with pEC-XK99E | 5 |
| EGS212 | LT-ΔfadE with pEG205 | 5 |
| EGS514 | BL21(DE3) with pEG513 | This study |
| EGS517 | BL21(DE3) with pEG516 | This study |
| EGS522 | DH1 ΔfadE ΔfadA | This study |
| EGS560 | EGS522 with pEG530 and pEC-XK99E | This study |
| EGS700 | EGS522 with pEG530 and pEG205 | This study |
| EGS735 | LT-ΔfadE with pEG705 | This study |
| EGS790 | LT-ΔfadE with pEG775 | This study |
| EGS860 | LT-ΔfadE with pEG855 | This study |
| EGS895 | EGS522 with pEG530 and pEG855 | This study |
| EGS975 | EGS522 with pEG955 and pEG855 | This study |
| EGS1015 | EGS522 with pEG530 and pEG990 | This study |
| EGS1080 | EGS522 with pEG530 and pEG1065 | This study |
| EGS1085 | EGS522 with pEG530 and pEG1070 | This study |
| EGS1090 | EGS522 with pEG530 and pEG1075 | This study |
| EGS1115 | EGS522 with pEG530 and pEG1101 | This study |
| EGS1120 | EGS522 with pEG530 and pEG1106 | This study |
| EGS1135 | DH1 with pEG1065 | This study |
| EGS1140 | DH1 with pEG1075 | This study |
| EGS1150 | LT-ΔfadE with pEG1065 | This study |
| EGS1155 | LT-ΔfadE with pEG1075 | This study |
| M. luteus strain ATCC 4698 | Wild type | ATCC |
| Plasmids | ||
| pEC-XK99E | Kmr; E. coli-Corynebacterium glutamicum shuttle expression vectors based on the medium-copy-no. plasmid pGA1 and containing the trc promoter | 14 |
| pKS1 | Cmr; p15a derivative containing ′tesA under the lacUV5 promoter | 26 |
| pKS104 | Ampr, ColE1 derivative with fadD (M335I), atfA under the lacUV5 promoter | 26 |
| pSKB3 | Kmr; a derivative of the expression vector pET-28a with the thrombin protease site replaced by a TEVb protease site | Burleya |
| pEG205 | Kmr; ∼1-kb fragment of Mlut_09310 (MlfabH) cloned into pEC-XK99E at EcoRI and XbaI sites | 5 |
| pEG513 | Kmr; ∼2.2-kb fragment of fadB (EcDH1_4135) cloned into pSKB3 at NdeI and SalI sites | This study |
| pEG516 | Kmr; ∼2.1-kb fragment of Mlut_11700 cloned into pSKB3 at NdeI and SalI sites | This study |
| pEG530 | Cmr; ∼2.1-kb fragment of Mlut_11700 and ∼2.2-kb fragment of fadB (EcDH1_4135) cloned downstream of the ′tesA gene in pKS1 by SLIC | This study |
| pEG705 | Kmr; QuikChange mutagenesis of MlfabH in pEG205 to the following residues: C123S, H275A, and N306A | This study |
| pEG775 | Kmr; ∼0.4-kb fragment of paaI (EcDH1_2249) cloned into pEC-XK99E at EcoRI and XbaI sites | This study |
| pEG855 | Kmr; ∼0.4-kb fragment of fadM (EcDH1_3166) cloned into pEC-XK99E at EcoRI and XbaI sites | This study |
| pEG955 | Cmr; ∼2.2-kb translational fusion of a leaderless maltose-binding protein gene (l-mbp) and the UcfatB1 gene, ∼2.1-kb fragment of Mlut_11700 and 2.2-kb fragment of fadB cloned into pKS1 (digested with MfeI and SalI to remove ′tesA) by SLIC | This study |
| pEG990 | Kmr; ∼1.7-kb of fadD M335I allele from pKS104 cloned downstream of fadM in pEG855 by SLIC | This study |
| pEG1065 | Kmr; ∼0.8-kb fragment of ShMKS1 and ∼0.6-kb fragment of ShMKS2 cloned into pEC-XK99E at BamHI and SalI sites by SLIC | This study |
| pEG1070 | Kmr; ∼0.8-kb fragment of ShMKS1 cloned into pEC-XK99E at BamHI and XbaI sites | This study |
| pEG1075 | Kmr; ∼0.6-kb fragment of ShMKS2 cloned into pEC-XK99E at BamHI and XbaI sites | This study |
| pEG1101 | Kmr; ∼ 0.9-kb fragment of Ptrc-ShMKS1 cloned downstream of fadM in pEG855 by SLIC | This study |
| pEG1106 | Kmr; ∼ 0.8-kb fragment of ShMKS1 cloned downstream of fadM in pEG855 by SLIC | This study |
| pEG1145 | Kmr; ∼1.2-kb fragment of hcaT (EcDH1_1132) into pEC-XK99E at EcoRI and XbaI sites | This study |
a
Stephen K. Burley.
b
TEV, tobacco etch virus.