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Pbad topo

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The PBAD-TOPO is a cloning vector used for the controlled expression of recombinant proteins in Escherichia coli. It features the araBAD promoter, which allows for tightly regulated, inducible expression of target genes upon the addition of arabinose to the culture medium. The vector also includes the TOPO cloning site, facilitating the rapid and efficient incorporation of PCR-amplified DNA fragments.

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Lab products found in correlation

Pgex 4t 1, by GE Healthcare (2 mentions) Pcoldii, by Takara Bio (2 mentions) Pacyc184, by New England Biolabs (1 mentions) Platinum pfx50 dna polymerase, by Thermo Fisher Scientific (1 mentions) Pgbkt7, by BD (1 mentions)

8 protocols using pbad topo

1

Plasmid Constructs for Vibrio Cholera

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Plasmid pBAD-lacZ is used as an empty vector control for experiments performed in V. cholerae and has been described previously [37 (link)]. Plasmid pBADTOPO-NrnB-VSVG was generated by PCR amplification of B. subtilis nrnB from pNrnB-VSVG [5 (link)] using primers NrnBOVERFW (5′- TAAGAGGAATAATAAATGTATCATTTATATTCACATAACG-3′), NrnBOVERRV (5′-GTCGACCTATTTTCCTAATCTATTCATTTC-3′), and cloning of this PCR product into pBADTOPO according to the manufacturer’s instructions (Life Technologies). Plasmid pNT01 carries sequences extending from −100 to +15 of the promoter associated with VCA0783 (pVCA0783) fused to the tR' terminator cloned into the HindIII and SalI sites of plasmid pACYC184 (New England Biolabs). Plasmids pNT02 and pNT03 are identical to pNT01 with the exception that the start site region associated with pVCA0783 carries the sequence C−1A+1 or C−1G+1, respectively. Plasmid pNT04 carries sequences extending from −100 to +15 of the promoter associated with VC1904 fused to the tR' terminator cloned into the HindIII and SalI sites of plasmid pACYC184. Plasmids pNT05 and pNT06 are identical to pNT04 with the exception that the start site region associated with pVC1904 carries the sequence C−1A+1 or C−1G+1, respectively.
Druzhinin S.Y., Tran N.T., Skalenko K.S., Goldman S.R., Knoblauch J.G., Dove S.L, & Nickels B.E. (2015). A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5′ RNA-seq. PLoS Genetics, 11(7), e1005348.
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2

Yeast-Two-Hybrid and In Vitro Binding Assays

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Fragments from PTB/PH domains (Suppl. Table 1), cytoplasmic domains of β integrins (ITGBs) and receptor tyrosine kinases (RTKs) (Suppl. Table 2) and NPXY-containing fragments of CCM1 (Suppl. Table 3) were amplified as described [5 (link)]. For Y2H, both GAL4 binding domain and activation domain fusion constructs were assembled, as described [5 (link),6 (link)]. For in vitro protein binding assays, PTB domain and NPXY motif constructs were assembled into pcDNA3.1/V5/HIS-TOPO and pcDNA4/HisMax-TOPO vectors (Invitrogen) for mammalian expression and pGEX-4T-1 (GE), pBAD-TOPO (Invitrogen), and pCOLDII (TaKaRa) vectors for bacterial expression [7 (link)].
In Y2H analysis, binding was determined by β-galactosidase liquid assay in selective (–His/–Ade) medium and is calculated as Miller units. To minimize intergroup measurement variation, the measurement of each transformant was normalized and converted to relative β-galactosidase activity (RBGA) as described [4 (link)–7 (link)].
Zhang J., Padarti A., Jiang X, & Abou-Fadel J. (2020). Redefining PTB domain into independently functional dual cores. Biochemical and biophysical research communications, 524(3), 595-607.
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3

Cloning and Expression of PTB and CCM1 Domains

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Candidate PTB/PH domains and full-length PTB-containing proteins (Suppl. Table 1) accompanied by full-length CCM1 (FLHK) and NPXY-containing fragments of CCM1, K2 (Residues 1–207), K5 (Residues 208–245), K8 (Residues 240–306) and K12 (Residues 208–306), were amplified with Platinum Pfx50 DNA Polymerase (Invitrogen) [15 (link)]. For yeast two-hybrid analysis, both GAL4 binding domain and activation domain fusion constructs were assembled by cloning amplified fragments into pGBKT7 and pGADT7 vectors, respectively (BD Clontech) [15 (link),16 (link)]. For in vitro protein binding assays, PTB domain and NPXY motif fusion constructs were assembled by cloning amplified fragments into pcDNA3.1/V5/HIS-TOPO and pcDNA4/HisMax-TOPO vectors (Invitrogen) for mammalian, and pGEX-4 T-1 (GE), pBAD-TOPO (Invitrogen) and pCOLDII (TaKaRa) vectors for bacterial expression systems.
Zhang J., Dubey P., Padarti A., Zhang A., Patel R., Patel V., Cistola D, & Badr A. (2017). Novel functions of CCM1 delimit the relationship of PTB/PH domains. Biochimica et biophysica acta. Proteins and proteomics, 1865(10), 1274-1286.
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4

Cloning toxRS from V. vulnificus

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A 1431-bp fragment of toxRS was amplified by PCR using primers pBAD_toxRS_F and pBAD_toxRS_R using the genomic DNA of V. vulnificus as a template. The resulting product was cloned into the pBAD-TOPO (Invitrogen, Carlsbad, CA) vector to generate pBAD-toxRS.
Park N.Y., Kim I.H., Wen Y., Lee K.W., Lee S., Kim J.A., Jung K.H., Lee K.H, & Kim K.S. (2019). Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus. Scientific Reports, 9, 20135.
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5

Construction of pBAD-hns and pBBR12-hns-ara Plasmids

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The 408-bp DNA fragment of the hns gene was amplified using primers BAD-hns-F and BAD-hns-R. The resulting fragment was cloned into pBAD-TOPO (Invitrogen, Thermo Fisher Scientific Inc., MA) to generate pBAD-hns. A 745-bp DNA fragment, including the ara promoter region fused to the upstream of promoter-less leuO, was amplified with primers pBAD-F and pBAD-R, and cloned into the EcoRI-digested pBBR1-MCS2 vector [18 (link)] using the In-fusion HD cloning kit (Clonetech Laboratories, TaKaRa Bio, Inc., Shiga, Japan) to generate pBBR12-hns-ara.
Park N.Y., Lee K.W, & Kim K.S. (2020). H-NS Silences Gene Expression of LeuO, the Master Regulator of the Cyclic(Phe-Pro)-dependent Signal Pathway, in Vibrio vulnificus. Journal of Microbiology and Biotechnology, 30(6), 830-838.
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6

Construct leuO Promoter and Expression Plasmids

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The 1,312-bp DNA fragment comprising the promoter region and the coding region of leuO was amplified by PCR using primers leuO_comP_F_xhoI and leuO_comP_R_kpnI. The resulting product was cloned into XhoI- and KpnI-digested pBBR1-MCS2 (56 (link)) to construct pBBR12-leuO. The 957-bp DNA fragment of the leuO gene was amplified using primers BAD-leuOF and BAD-leuOB. The resulting fragment was cloned into pBAD-TOPO (Invitrogen, Thermo Fisher Scientific Inc., MA) to generate pBAD-leuO. A 2,700-bp DNA fragment including the ara promoter region fused to the promoterless leuO was amplified with primers BBR12-bad-leuOF and BBR12-bad-leuOB and cloned into the EcoRI-digested pBBR1-MCS2 vector (56 (link)) using the In-fusion HD cloning kit to generate pBBR12-leuO-ara.
Kim I.H., Kim S.Y., Park N.Y., Wen Y., Lee K.W., Yoon S.Y., Jie H., Lee K.H, & Kim K.S. (2018). Cyclo-(l-Phe-l-Pro), a Quorum-Sensing Signal of Vibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress. Infection and Immunity, 86(9), e00932-17.
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7

Complementation Assay for BcTSA Activity

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The complementation assays were performed essentially as described with some modifications (Zhang et al., 2008 (link)). The E. coli ΔtrpA mutant strain CGSC 9129, which contains an interruption/deletion of the trpA gene, was purchased from Yale University’s E. coli Genetic Stock Center. The ORF of BcTSA was cloned in pBAD-TOPO (Invitrogen, Carlsbad, CA, USA) expression vector for functional expression in E. coli using specific primers specified in Table S1 according to the manufacturer’s instructions. The untransformed E. coli mutant strain was cultured in LB medium with kanamycin at a concentration of 50 mg/mL. All strains were cultivated on M9 basic medium, supplemented with 1M MgSO4, 1M CaCl2, 20% glucose, glycerol, 100 mg/mL of ampicillin, and 20% arabinoses for inducible expression of BcTSA, then cultured at 37°C in the dark for 2 d. The untransformed strains or transformed strains containing the pBAD-TOPO vector serve as a negative control. The medium supplemented with 100 mg/mL tryptophan was used as a positive control.
Guo Z., Chen J., Lv Z., Huang Y., Tan H., Zhang L, & Diao Y. (2023). Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia. Frontiers in Plant Science, 14, 1174582.
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8

Screening Lipase-Producing Bacterial Strains

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Lipase-producing strains of Burkholderia cepacia and Pseudomonas aeruginosa were used as positive controls, and Escherichia coli DH5α was used as the negative control. These strains are part of the "Banco de Cepas y Genes del Instituto de Biotecnología de la Universidad Nacional de Colombia". E. coli TOP10 and plasmid pBAD-TOPO (Invitrogen) were used for cloning/expression assays. Luria Bertani agar (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, 15 g/L bacteriological agar) supplemented with tributyrin 1 % v/v and 0.2 ml/L triton X-100 (emulsifier) was used for lipase screening. To confirm lipolytic activity, Tween 20 (10 g/L peptone, 5 g/L NaCl, 15 g/L bacteriological agar, 1.1 g/L CaCl2 and 10 ml/L Tween 20) and Rhodamine-Olive Oil-Agar (8 g/L nutrient broth, 4 g/L NaCl, 15 g/L bacteriological agar, 10 ml/L Olive Oil and 10 ml/L Rhodamine B) were also used. Some culture media contained 0.1 mg/L ampicillin antibiotic.
Ruiz-Toquica J.S., González N.C., & Castaño D.M. (2020). Two possible candidate enzymes from Ulva lactuca-associated epiphytic bacteria obtained through PCR and functional evaluation. Universitas Scientiarum, 25(2), 247-275.
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