pTDpelB-NTwinStrep
(Plasmid #45940)

• Purpose: (Empty Backbone) use to create N-terminal PelB-Twin-Strep-tag fusion proteins or dual tagged fusion with N-terminal PelB-Twin-Strep-tag and C-terminal 6x His-tag for periplasmic translocation via PelB signal sequence
• Depositing Lab: Thorben Dammeyer
• Publication: Dammeyer et al Microb Cell Fact. 2013 May 20;12(1):49.

Backbone

• Vector backbone: pSEVA424
• Backbone manufacturer: SEVA (de Lorenzo Lab)
• Modifications to backbone: Insertion of a synthetic MfeI/HindIII fragment providing a new, customized MCS with RBS, PelB leader sequence and N-terminal Twin-Strep-tag in the EcoRI/HindIII sites of pSEVA424
• Vector type: Bacterial Expression
• Promoter: lacIq-Ptrc
• Tags / Fusion Proteins:
  • PelB export signal (N terminal on backbone)
  • Twin-Strep-tag (N terminal on backbone)
  • HIS-6-tag (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Streptomycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For growth in P.putida KT2440 (30C); on plates use spectinomycin in addition.
• Copy number: Low Copy

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: 5′-TGTGTGGAATTGTGAGCGG-3′
• 3′ sequencing primer: 5′-ACTTTGTTTTAGGGCGACTG-3′

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest for 45940

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This plasmid was tested in the Gram-negative soil bacterium Pseudomonas putida KT2440 and Escherichia coli K12 and is especially suited for protein production, affinity purification, protein complex copurification with SPINE (Strep Protein Interaction Experiments) or (co-)localization studies. Due to the broad host range of the RK2 origin of replication, the plasmid facilitates experimental verification of hypothetical proteins and protein production yield assessment in different expression hosts possibly including new isolates.

The Supplementary Table S1 in the following publication lists approximately 30 strains in which the RK2 origin of replication should be functional. Silva-Rocha et al., The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Research 2013, 41:D666-675. http://nar.oxfordjournals.org/content/41/D1/D666.long