Strains |
E. coli DH5α |
Cloning strain |
|
V. atypica OK5 |
Wild type |
(Liu et al., 2012) |
Δecf3 |
OK5 ecf3 deletion mutant |
This work |
Plasmids |
pBST |
Suicide vector of V. atypica, the beta-lactamase gene in pBluescript II KS (+) was replaced by tetM
|
(Liu et al., 2012) |
pBST-Pmdh-pheS*
|
Carrier plasmid for markerless deletion of any gene |
This work |
pBST-Pmdh-pheS*-ecf3
|
Carrier plasmid for ecf3 markerless deletion |
This work |
Primers |
Sequence (5′ to 3′) |
Purpose |
pheS-F |
ATGGAACAAGAATTACAACGCATA |
pheS amplification |
pheS-R-EcoRI |
CGGAATTCCTAAAATTGTTCCAAGAAACGGATATCA |
pheS amplification |
Pmdh-F-XhoI |
CCGCTCGAGATACATACATCACTATATCTGTAACA |
mdh promoter amplification |
Pmdh-R |
TATGCGTTGTAATTCTTGTTCCATTGTTAAAACCTCTTTTCAGAAAATATGTA |
mdh promoter amplification |
pheSm-F |
CCAAAACCTTTCACCTTATTAGGATCA |
Site-directed mutation of pheS
|
pheSm-R |
TTTTGGTATGGGCGTAGAACGTA |
Site-directed mutation of pheS
|
ecf3-KO-up-F |
CGGGATCCGAAAAGAGTTTTTTGTGTGA |
ecf3 deletion |
ecf3-KO-up-R |
TAAAAAATATTTTAGATTTTTAAAAGATTCGTTCCTTTCTGCCTA |
ecf3 deletion |
ecf3-KO-down-F |
TAGGCAGAAAGGAACGAATCTTTTAAAAATCTAAAATATTTTTTA |
ecf3 deletion |
ecf3-KO-down-R |
GCTCTAGAGTATGCCGATATTATAGGCTGCA |
ecf3 deletion |