TABLE 3.
Plasmids used in the deletion assay
| Plasmid | Descriptiona |
|---|---|
| pSU18 | oriP15A Cmr (3) |
| pSU18Δ | Deletion of the EcoRI/NheI (233-bp) fragment of pSU18 carrying the 3′ end of lacI and the lacZ promoter and replacement by an EcoRV restriction site (see text) |
| pMBG18R | oriColE1 Apr, mp18 MCS containing additional in-frame BglII and MfeI sites (Demarre et al., in press). |
| pNOT218 | oriColE1 Apr (33) |
| pBAD18 | oriColE1 Apr, expression vector (12) |
| pSU38::ORF1-cat | pSU38::VCR1-ΔORF1::cat-VCR2 (26) |
| pTRC99A | oriColE1 Apr, expression vector (Pharmacia) |
| pTZ18R::dfrB1 | oriColE1dfrB1 Tpr Apr (25) |
| p734 | intI1 carrying EcoRI/HindIII fragment from p112 (33) cloned in pBAD18 |
| p995 | VchintIA EcoRI/SphI fragment from pVC3 (33) cloned in pBAD18 |
| Synthetic cassette assembly | |
| pSU18Δ::lacIq | 1,254-bp EcoRI/BamHI lacIq PCR fragment (IQA and IQB) amplified from pTRC99A in pSU18Δ |
| p452 | 200-bp EcoRI/BamHI VCR1 PCR fragment (REV-48 and C1B) amplified from pSU38::ORF1-cat cloned in EcoRI/BamHI-digested pSU18Δ |
| p453 | 200-bp EcoRI/BamHI VCR2 PCR fragment (2A and 2B) amplified from pSU38::ORF1-cat in EcoRI/BamHI-digested pSU18Δ |
| p457 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p452 digested by MfeI/HindIII; pSU18Δ::VCR1-lacIq |
| p470 | 200-bp EcoRI/BamHI VCR2 fragment from p453 in pSU18Δ::lacIq |
| p674b | 188-bp BglII/HindIII VCR2 fragment from p453 in p457; pSU18Δ::VCR1-lacIq-VCR2 |
| p1011 | 79-bp BglII/XbaI attCaadA7 fragment obtained by annealing between the AADA7.1 and AADA7.2 primers, filling with the Pfu DNA polymerase, digestion, and cloning in pMBG18R |
| p1182b | 99-bp BglII/HindIII attCaadA7 fragment from p1011 in p674; pSU18Δ::VCR1-lacIq-attCaadA7 |
| p2899 | 86-bp EcoRI/BamHI attCorfA fragment obtained by annealing between the orfA4 and orfA5 primers, filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p2969 | 153-bp EcoRI/BamHI VCR2 PCR fragment (VCR2RI and orfA3) amplified from p453 in pSU18Δ |
| p2978 | 148-bp EcoRI/BamHI VCR2 PCR fragment (VCR2RI and dfrA1III) amplified from p453 in pSU18Δ |
| p2981 | 116-bp EcoRI/BamHI attCdfrA1 fragment obtained by annealing between the dfrA1I and dfrA1II primers, filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p2984 | 91-bp EcoRI/BamHI attCorfA fragment obtained by annealing between the orfA6 and orfA7 primers, filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p3012 | 148-bp EcoRI/BamHI VCR2 PCR fragment (VCR2RI and ereA1a) amplified from p453 in pSU18Δ |
| p3015 | 73-bp EcoRI/BamHI attCereA fragment obtained by annealing between the ereA1b and ereA1c primers, filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p3019 | 88-bp EcoRI/BamHI attCorfA PCR fragment (orfA8 and orfA9) amplified from p2899 in pSU18Δ |
| p3028 | 166-bp EcoRI/BamHI VCR2 fragment from p453 in pSU18Δ |
| p3074 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p3012 digested by MfeI/HindIII |
| p3075 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p3019 digested by MfeI/HindIII |
| p3076 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p2969 digested by MfeI/HindIII |
| p3077 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p2978 digested by MfeI/HindIII |
| p3078b | 97-bp BglII/HindIII attCereA fragment from p3015 in p3074; pSU18Δ::VCR2-lacIq-attCereA |
| p3079b | 190-bp BglII/HindIII VCR2 fragment from p3028 in p3075; pSU18Δ::attCorfA-lacIq-VCR2 |
| p3080b | 115-bp BglII/HindIII attCorfA fragment from p2984 in p3076; pSU18Δ::VCR2-lacIq-attCorfA |
| p3113b | 140-bp BglII/HindIII attCdfrA1 fragment from p2981 in p3077; pSU18Δ::VCR2-lacIq-attCdfrA1 |
| p3212 | 76-bp EcoRI/BamHI attCaadB fragment obtained by annealing between the aadB-1 and aadB-2 primers filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p3214 | 151-bp EcoRI/BamHI VCR2 PCR fragment (VCR2RI and aadB-3) amplified from p453 in pSU18Δ |
| p3230 | 1,275-bp EcoRI/HindIII lacIq fragment from pSU18Δ::lacIq in p3214 digested by MfeI/HindIII |
| p3233 | 77-bp EcoRI/BamHI attCaadB fragment obtained by annealing between the aadB-4 and aadB-5 primers, filling with the Pfu DNA polymerase, digestion, and cloning in pSU18Δ |
| p3235b | 100-bp BglII/HindIII attCaadB fragment from p3212 in p3230; pSU18Δ::VCR2-lacIq-attCaadB |
| p3245 | 1275-bp EcoRI/HindIII laclq fragment from pSU18Δ::lacIq in p3233 digested by MfeI/HindIII |
| p3249b | 99-bp BglII/HindIII attCaadA7 fragment from p1182 in p3245; pSU18Δ::attCaadB-lacIq-attCaadA7 |
| VCR mutant construction | |
| p440 | 214-bp EcoRI/BamHI VCR1 PCR fragment (REV-48 and C1B3) from pSU38::ORF1-cat in pSU18Δ |
| p441 | 214-bp EcoRI/BamHI VCR1 PCR fragment (REV-48/C1B2) amplified from pSU38::ORF1-cat in pSU18Δ |
| p454 | 168-bp BglII/BamHI VCR2 PCR fragment (2A2 and 2B) amplified from p453 in pSU18Δ |
| p471 | 168-bp BglII/BamHI VCR2 PCR fragment (2A2 and 2B) from p454 in pSU18Δ::lacIq |
| p675b | 162-bp BglII/BamHI VCR2 PCR fragment (2A4 and 2B) amplified from p453 in p457 |
| p735b | 168-bp BglII/BamHI VCR2 PCR fragment (2A2 and 2B) from p454 in p457 |
| p745b | 1,425-bp EcoRI/BamHI lacIq-VCR2 PCR fragment (2A2 and 2B) from p471 in p441 digested by MfeI/HindIII |
| p746b | 1,425-bp EcoRI/HindIII lacIq-VCR2 PCR fragment from p470 in p440 digested by MfeI/HindIII |
| p1046b | 141-bp BglII/PstI VCR2 PCR fragment (I-STU and WT-CS) amplified from p453 in p457 |
| p1047b | 141-bp BglII/PstI VCR2 PCR fragment (I-SNA and WT-CS) amplified from p453 in p457 |
| p1048b | 141-bp BglII/PstI VCR2 PCR fragment (I-ACL and WT-CS) amplified from p453 in p457 |
| p1049b | 141-bp BglII/PstI VCR2 PCR fragment (WT-ICS and C-BSS) amplified from p453 in p457 |
| p1050b | 141-bp BglII/PstI VCR2 PCR fragment (WT-ICS and C-BSI) amplified from p453 in p457 |
| p1051b | 141-bp BglII/PstI VCR2 PCR fragment (WT-ICS and C-ACL) amplified from p453 in p457 |
| p1653b | 148-bp BglII/HindIII VCR2 PCR fragment (REV-48 and VCR2C-T) amplified from p453 in p457 |
| p1676b | 187-bp BglII/HindIII VCR2 PCR fragment (IVCR2A-G and FD-40) amplified from p453 in p457 |
| p2331b | 1,425-bp EcoRI/HindIII lacIq-VCR2 from p470 in p441 digested by MfeI/HindIII |
| Deletion reporter construction | |
| pNOT::Ptac | 109-bp Ptac promoter PCR fragment (PLAC1 and PLAC2) in pNOT218 |
| pNOTΔ::Ptac | Deletion of the lacZ promoter by inverse PCR (PNOTΔ and PTRC1), EcoRI digestion, and religation |
| pNOTΔ::Ptac-dfrB1 | 318-bp SacI and BamHI fragment from pTZ18R::dfrB1 in pNOTΔ::Ptac |
a
The numbers in parentheses are reference numbers.
b
Plasmid corresponding to a final construct, synthetic cassette, and mutant, which have been tested in the deletion assay.