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
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||45940||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerSEVA (de Lorenzo Lab)
Modifications to backboneInsertion 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 typeBacterial Expression
- Promoter lacIq-Ptrc
/ 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
Growth instructionsFor growth in P.putida KT2440 (30C); on plates use spectinomycin in addition.
Copy numberLow Copy
- Cloning method Restriction Enzyme
- 5′ sequencing primer 5′-TGTGTGGAATTGTGAGCGG-3′
- 3′ sequencing primer 5′-ACTTTGTTTTAGGGCGACTG-3′ (Common Sequencing Primers)
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
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pTDpelB-NTwinStrep was a gift from Thorben Dammeyer (Addgene plasmid # 45940 ; http://n2t.net/addgene:45940 ; RRID:Addgene_45940)
For your References section:Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein. Dammeyer T, Timmis KN, Tinnefeld P. Microb Cell Fact. 2013 May 20;12(1):49. 10.1186/1475-2859-12-49 PubMed 23687945