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Bacterial

OG162 - pSF-T7-NH2-OmpA-SP1

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This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Product Code: OG162

Size (bp): 3936

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322   

Copy Number: 500-700 copies per cell

Promoter: T7 bacteriophage Promoter

Purpose: This vector allows transcription of un-polyadenylated and un-capped RNA using the T7 bacteriophage  polymerase. This vector also contains the Escherichia coli (E.coli) outer membrane protein A (OmpA) secretory signal peptide. When positioned at the N-terminus of a protein it should direct the protein to be     secreted out of the bacterial cytosol and into the periplasmic space. The OmpA secretory tag is 21 amino acids in length. The signal peptide protein sequence is MKKTAIAIAVALAGFATVAQA. Cleavage occurs after the final Alanine  residue. The peptide tag will be cleaved the mature protein during export.

This technique can be used to increase di-sulphide bond formation and also reduces proteolytic degradation. This method also reduces contamination from endogenous proteins because very few E.coli proteins are secreted. A sub fraction of the protein secreted into the periplasmic space can also extracted from the growth media. It is still unclear why this occurs but it believed to reflect increased permeability of the outer membrane, particularly during extended culture periods.

Secretion of proteins in E.coli can sometimes be difficult. Potential issues include protein toxicity within the periplasm, variable secretion efficiency, formation of inclusion bodies within the cytoplasm when using strong promoters, incorrect dis-sulphide formation. Background to bacterial secretion systems, including possible solutions to these problems can be found in Choi et al 2004, Applied Microbiology and Biotechnology, 64 (625-635).

All SnapFastTM vectors (except those in the terminators category on our website) will contain a T7 terminator downstream of the multiple cloning site. This will enable transcription termination.    

Frame: For reference, the OmpA coding sequence is in frame with the ATG start codon within the NciI restriction site.

Inserting a Gene: Each restriction site in this vector has a purpose and allows the insertion of specific pre-designed DNA sequences. To insert a gene, we recommend using any sites between (and including) the HindIII and XbaI sites. By doing this you will be able to insert most of our other products upstream, or downstream, of your gene. Click here to read more about the purpose of each restriction site.

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

Transcription Termination: This plasmid contains three alternative transcription terminators for mammalian, bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.
                                    
Quality Validation: This vector has been demonstrated to secrete reporter genes in T7 expressing bacterial cells. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned within the XbaI site.

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The sequences and details of these primers are available through our website.

Genetic Modifications to standard parts:

  • KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.

Restriction site notes:

  • Bgl2, KpnI, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, and KanR, respectively.
  • BsgI and BseRI recognise non-palindromic DNA sequences and cleave upstream of their recognition sites, within the stop codon that is found inside the XbaI restriction site.