Table 1 Strains, plasmids and primers used in cloning and expression of bisd A and bisd B and knocking out of bisd B
Strains, Plasmids and Primers | Description | Source |
|---|---|---|
Pseudomonas putida strains | ||
YC-AE1 | Wild-type bisphenol A degrader | [21] |
YC-AE1ΔbisdB | YC-AE1 mutant with bisdB gene replaced with kanamycin resistance gene | This study |
E. coli strains | ||
Trans1-T1 | F-φ80(lacZ)ΔM15ΔlacX74hsdR(rk−,mk+)ΔrecA1398endA1tonA | TransGen |
BL21(DE3) | Host strain for expression vextors; F− ompT hsdSB(rB− mB−) gal dcm(DE3) | Tiangen |
SM10λpir | Donor strain for conjugation, thi thr leu tonA lacY supE recA∷RP4-2-Tc∷Mu | Zomanbio |
Plasmids | ||
pET32a( +) | Expression vector; Ampr | Novagen |
pEX18Tc | Gene knockout vector, oriT, sacB, Tcr | Miaolingbio |
pET32a-bisdB | pET-28a( +) derivative carrying bisdB | this study |
pET32a-bisdAB | pET-28a( +) derivative carrying bisdA and bisdB | this study |
pEX18Tc-bisdB | pEX18Tc derivative carrying bisdB | this study |
Primers Sequence (5’ → 3’) | ||
bisdB-F | GCGCGAGCTCATGAACCCTCAGACACTGC | this study |
bisdB-R | GCGCAAGCTTGTTTTTGTCCCAGACCAGC | this study |
bisdAB-F | GCGCGAGCTCATGCCTCATATCCAAGTGACT | this study |
bisdAB-R | GCGCAAGCTTGTTTTTGTCCCAGACCAGC | this study |
bisdBup-F | TAAAACGACGGCCAGTGCCATTACTCAGCGAGCCGCGTT | this study |
bisdBup-R | TCCCGTTGAATATGGCTCATGTTCGGATTCCCGCTCATTTTCG | this study |
kan-F | ATGAGCCATATTCAACGGGAAACGT | this study |
kan-R | TTAGAAAAACTCATCGAGCATCAAATGAAAC | this study |
bisdBdown-F | GATGCTCGATGAGTTTTTCTAAGCCGGGCTTTCAAGTACCTGAGCAGATG | this study |
bisdBdown-R | ACCATGATTACGAATTCGAGCTGCCATCGACTGCGCAGACATG | this study |