pMSP3535VA
(Plasmid #46887)

• Purpose: (Empty Backbone) Gram-Positive Bacterial Shuttle Vector for Nisin-Controlled Inducible Expression
• Depositing Lab: Gary Dunny
• Publication: Bryan et al Plasmid. 2000 Sep;44(2):183-90.

Backbone

• Vector backbone: pMSP3535VA
• Backbone size (bp): 8278
• Modifications to backbone: See associated article for detailed construction information
• Vector type: Bacterial Expression ; Nisin-inducible expression; Gram-positive Bacterial Shuttle Vector
• Promoter: PnisA

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): Stbl3
• Growth instructions: For E. coli, grow on Luria Broth (LB) agar containing 50 μg/mL kanamycin. For E. faecalis, grow on Brain Heart Infusion (BHI) medium containing 1000 μg/mL kanamycin.
• Copy number: Low Copy

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: Unknown
• 3′ sequencing primer: T7; pBR322ori-F (5'-GGGAAACGCCTGGTATCTTT-3')

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest
• A portion of this plasmid was derived from a plasmid made by: The pNisA promoter and the nisin-sensing genes come from the Netherlands Dairy Research Institute (NIZO) (de Ruyter, Kuipers, and de Vos, 1996. PMID: 8837421)
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

This plasmid utilizes the NIsin Controlled Expression (NICE) system. NICE® is a registered trademark of NIZO food research BV, P.O.Box 20, 6710 BA Ede, The Netherlands.

pMSP3535VA contains the Nisin-inducible PnisA promoter, and the pVA380-1 replicon for expression in gram-positive bacteria.

For protein expression in E. faecalis, induce with 10-25 ng/mL nisin (Sigma, N-5764). See associated article for instructions on how to make nisin solution.

Note: Nucleotide sequence encoding the signal sequence and one-third of the mature NisA (nisin) peptide remains associated with the PnisA promoter. The depositing laboratory recommends adding of a Stop codon to the 5' end of inserted sequences to avoid unintentional gene fusions.

Note: The theoretical full sequence uploaded here is NOT fully accurate - the NheI site at position 4414 has been lost, along with approximately 500bp of sequence in this region (compare the depositor's uploaded maps). The actual size of this plasmid is 8278 as listed here. The depositing lab has not resequenced the region in question, but the discrepancy does not affect plasmid function.

Note: This plasmid has been shown to accumulate deletions when replicated in E. coli (or gram-negative bacteria). Screening transformants by restriction digest is recommended.

Addgene's quality control sequencing finds discrepancies with the depositor's provided sequence, but they are not thought to affect plasmid function.