Table 1.
Expression vectors and oligonucleotides designed and used in this study
| Plasmid | Description | Reference |
|---|---|---|
| pBP99 | Vector encoding a tetracycline-responsive SAMY expression unit (PhCMV*-1-SAMY-pA). pCF59 was restricted with BprPI/EcoRV and religated. | unpublished |
| pBP100 | Vector encoding an erythromycin-responsive SAMY expression unit (PETR3-SAMY-pA). | 18 |
| pcDNA3.1 | Commercial cloning vector containing a constitutive promoter (PhCMV). | Invitrogen |
| pCF59 | Vector encoding a PPIR-driven SAMY expression unit (PhCMV*-1-pA-IRES-PPIR-SAMY-pA). SAMY was excised from pSS158 using SpeI/BglII and ligated into pMF187 (SpeI/BglII). | unpublished |
| pCK9 | Vector encoding a PUREX8-driven SEAP expression unit (PUREX8-SEAP-pA). | 15 |
| pCK73 | Vector encoding a uric acid-responsive SEAP expression unit, driven by a size-reduced PhCMV (PhCMV-8xhucO-SEAP-pA). Size reduced PhCMV was amplified using OCK72: 5′- ccgctcgaggaagatctCGCGTTGACATTGATTATTGAC-3′ and OCK74: 5′- gcgacgtcccaagcttGCTTATATAGACCTC CCACC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with XhoI/HindIII and ligated into pCK9 (XhoI/HindIII). | 15 |
| pCK188 | Constitutive VanA4 expression vector (PSV40- VanA4-pA; VanA4, VanR-KRAB). VanR was PCR-amplified from pMG250 using OCK190: 5′- ggaattccaccATGGACATGCCGCGCATAAAG-3’ and OCK191: 5′ ggcgacgcgtaGTCGGCGCGAATGCTCCAC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with EcoRI/XbaI and ligated into pWW43 (EcoRI/BssHII). | This work |
| pCK189 | Constitutive VanA4 expression vector (PhCMV-VanA4-pA). VanR was PCR-amplified from pCK188 using OCK190: 5′- ggaattccaccATGGACATGCCGCGCATAAAG-3′ and OCK192: 5′- gctctagaCTTACCAGAGATCATTCCTTGC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with EcoRI/XbaI and ligated into pcDNA3.1 (EcoRI/XbaI). | This work |
| pCK191 | Vector encoding a PvanON8-driven SEAP expression unit (PVanON8-SEAP-pA; PvanON8, PhCMV-VanO8). Oligonucleotides OCK193: 5′-agcttATTGGATCCAATGCATTGG ATCCAATGGATTGGATCCAATCGATTGGATCCAATgatatcATTGGATCCAATGCATTGGATCCAATGGATTGGATCCAATCGATTGGATCCAATg-3′ and OCK194: 5′- aattcATTGGATCCAATCGATTGGATCC AATCCATTGGATCCAATGCATTGGATCCAATgatatcATTGGATCCAATCGATTGGATCCAATCCATTGGATCCAATGCATTGGATCCAATa-3′ (lower case italics, restriction sites; upper case, 8xVanO) were annealed and ligated directly into pCK73 using HindIII/EcoRI. | This work |
| pd2EYFP | Mammalian d2EYFP expression vector | Clontech |
| pMF187 | Dual-regulated expression vector (PhCMV*-1-MCSI-IRES-MCSII-pAI-PPIR-MCSIII-pAII). | 68 |
| pMG10 | Vector encoding a PTtgR1-driven SEAP expression unit (PTtgR1-SEAP-pA; PTtgR1, OTtgR-0bp-PhCMVmin). | 28 |
| pMG18 | Constitutive TtgA2 expression vector (PSV40-TtgA2-pA). | 28 |
| pMG19 | Constitutive TtgA3 expression vector (PSV40-TtgA3-pA). | 28 |
| pMG20 | Vector encoding a PTtgR2-driven SEAP expression unit (PTtgR2-SEAP-pA; PTtgR2, OTtgR-2bp-PhCMVmin). | 28 |
| pMG21 | Vector encoding a PTtgR3-driven SEAP expression unit (PTtgR3-SEAP-pA; PTtgR3, OTtgR-4bp-PhCMVmin). | 28 |
| pMG22 | Vector encoding a PTtgR4-driven SEAP expression unit (PTtgR4-SEAP-pA; PTtgR4, OTtgR-6bp-PhCMVmin). | 28 |
| pMG23 | Vector encoding a PTtgR5-driven SEAP expression unit (PTtgR5-SEAP-pA; PTtgR5, OTtgR-8bp-PhCMVmin). | 28 |
| pMG24 | Vector encoding a PTtgR6-driven expression unit (PTtgR6-SEAP-pA; PTtgR6, OTtgR-10bp-PhCMVmin). | 28 |
| pMG250 | Constitutive VanA1 expression vector (PSV40-VanA1-pA; VanA1, VanR-VP16). VanR was PCR-amplified from C. crescentus genomic DNA using ODF150: 5′-cggaattccaccATGGACATGCCGCGCATAAAGCCGGGC-3′ and ODF151: 5′- gtacgacgcgtggctgtacgcggaGTCGGCGCGAATGCT CCACGCCGCGC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with EcoRI/MluI and ligated into pWW35 (EcoRI/BssHII). | This work |
| pMG252 | Vector encoding a P1VanO2-driven SEAP expression unit (P1VanO2-SEAP-pA; P1VanO2, VanO2-0bp-PhCMVmin). VanO2 was created by annealing Oligos OMG65 (5′-phosphate-tcgacgtcaattcgcgaATTGGATCCAATagc gctATTGGATCCAATgatatcggacctgca-3′; lower case italics, restriction sites; upper case italics, VanO) and OMG66 (5′-phosphate-gcagttaagc gctTAACCTAGGTTAtcgcgaTAACCTAGGTTActatagcctgg-3′; lower case italics, restriction sites; upper case italics, VanO), and was directly ligated into SalI/SbfI-digested pWW1088 thereby resulting in P1VanO2. | This work |
| pMG256 | Constitutive VanA2 expression vector (PSV40-VanA2-pA; VanA2, VanR-p65). VanR was PCR-amplified from C. crescentus genomic DNA using ODF150: 5′-cggaattccaccATGGACATGCCGCGCATAAAGCCGGGC-3′ and ODF151: 5′- gtacgacgcgtggctgtacgcggaGTCGGCGCGAATGCT CCACGCCGCGC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with EcoRI/MluI and ligated into pMG18 (EcoRI/BssHII). | This work |
| pMG257 | Constitutive VanA3 expression vector (PSV40-VanA3-pA; VanA3, VanR-E2F4). VanR was PCR-amplified from C. crescentus genomic DNA using ODF150: 5′-cggaattccaccATGGACATGCCGCGCATAAAGCCGGGC-3′ and ODF151: 5′- gtacgacgcgtggctgtacgcggaGTCGGCGCGAATGCT CCACGCCGCGC-3′ (lower case italics, restriction sites; upper case italics, annealing), digested with EcoRI/MluI and ligated into pMG19 (EcoRI/BssHII). | This work |
| pMG262 | Vector encoding a P1VanO1-driven SEAP expression unit (P1VanO1-SEAP-pA; P1VanO1, VanO1-0bp-PhCMVmin). pMG252 was digested using either NruI/HindIII or Eco47III/HindIII. The resulting fragments were ligated to result in pMG262 harboring one VanO-operator element. | This work |
| pMG263 | Vector encoding a P1VanO3-driven SEAP expression unit (P1VanO3-SEAP-pA; P1VanO3, VanO3-0bp-PhCMVmin). pMG252 was digested using either EcoRV/HindIII or Eco47III/HindIII. The resulting fragments were ligated to result in pMG263 harboring three VanO-operator elements. | This work |
| pMG264 | Vector encoding a P1VanO4-driven SEAP expression unit (P1VanO4-SEAP-pA; P1VanO4, VanO4-0bp-PhCMVmin). pMG252 was digested using either EcoRV/HindIII or NruI/HindIII. The resulting fragments were ligated to result in pMG264 harboring four VanO-operator elements. | This work |
| pMG265 | Vector encoding a P2VanO2-driven SEAP expression unit (P2VanO2-SEAP-pA; P2VanO2, VanO2-2bp-PhCMVmin). 2bp-PhCMVmin-SEAP was excised from pMG20 (SbfI/XhoI) and ligated into pMG252 (SbfI/XhoI). | This work |
| pMG266 | Vector encoding a P3VanO2-driven SEAP expression unit (P3VanO2-SEAP-pA; P3VanO2, VanO2-4bp-PhCMVmin). 4bp-PhCMVmin-SEAP was excised from pMG21 (SbfI/XhoI) and ligated into pMG252 (SbfI/XhoI). | This work |
| pMG267 | Vector encoding a P4VanO2-driven SEAP expression unit (P4VanO2-SEAP-pA; P4VanO2, VanO2-6bp-PhCMVmin). 6bp-PhCMVmin-SEAP was excised from pMG22 (SbfI/XhoI) and ligated into pMG252 (SbfI/XhoI). | This work |
| pMG268 | Vector encoding a P5VanO2-driven SEAP expression unit (P5VanO2-SEAP-pA; P5VanO2, VanO2-8bp-PhCMVmin). 8bp-PhCMVmin-SEAP was excised from pMG23 (SbfI/XhoI) and ligated into pMG252 (SbfI/XhoI). | This work |
| pMG269 | Vector encoding a P6VanO2-driven SEAP expression unit (P6VanO2-SEAP-pA; P6VanO2, VanO2-10bp-PhCMVmin). 10bp-PhCMVmin-SEAP was excised from pMG24 (SbfI/XhoI) and ligated into pMG252 (SbfI/XhoI). | This work |
| pMG270 | Autoregulated vanillic acid-controlled SEAP expression vector (P1VanO2-SEAP-IRESPV-VanA1-pA). VanA1 was excised from pMG250 (SspI/NotI) and ligated into pMG252 (SspI/NotI). | This work |
| pPur | Selection vector conferring puromycin resistance. | Clontech |
| pSAM200 | Constitutive tTA expression vector (PSV40-tTA-pA). | 69 |
| pSEAP2-Control | Constitutive SEAP expression vector (PSV40-SEAP-pA). | Clontech |
| pSS158 | PhCMV-driven SAMY expression vector (PhCMV-SAMY-pA). | 50 |
| pSV2neo | Selection vector conferring neomycin resistance. | Clontech |
| pWW35 | Constitutive ET1 expression vector (PSV40-ET1-pA; ET1, E-VP16). | 18 |
| pWW43 | PSV40-driven expression vector encoding the macrolide-dependent transrepressor ET4: PSV40-ET4-pA; ET4, E-KRAB. | 18 |
d2EYFP, destabilized variant of the yellow fluorescent protein; E2F4, transactivation domain of the human E2F transcription factor 4; ET1, macrolide-dependent transactivator (E-VP16); ET4, macrolide dependent transrepressor (E-KRAB); ETR, operator specific for macrolide-dependent transactivators; IRESPV, polioviral internal ribosome entry site; KRAB, Human Kruppel-associated box protein; NF-κB, human transcription factor; OTtgR, TtgR-specific operator; p65, transactivation domain of NF-κB; pA, polyadenylation site; PETR3, macrolide-responsive promoter; PhCMV, human cytomegalovirus immediate early promoter; PhCMVmin, minimal PhCMV; PhCMV*-1, tetracycline-responsive promoter; PSV40, simian virus 40 promoter; PTtgR1-6, phloretin-responsive promoters containing different spacers between OTtgR and PhCMVmin; PUREX8, uric acid- responsive promoter containing 8 hucO-operator sites in 3′ of a PCMV promoter; P1-6VanO2, vanillic acid-responsive promoters containing different spacers between VanO and PhCMVmin; P1VanO1-4, vanillic acid-responsive promoters harboring 1, 2, 3 or 4 VanO-operator repeats in front of PhCMVmin; SEAP, human placental secreted alkaline phosphatase; SAMY, Bacillus stearothermophilus-derived secreted α-amylase; TtgR, repressor of the Pseudomonas putida DOT-T1E ABC multi-drug efflux pump; TtgA2, phloretin-dependent transactivator (TtgR-p65); TtgA3, phloretin-dependent transactivator (TtgR-E2F4); VanAB, gene cluster within C. crescentus that plays a role within the vanillic acid metabolism; VanO, VanR specific operator; VanR, repressor of the C. crescentus VanAB gene cluster; VP16, Herpes simplex virus-derived transactivation domain.