pZE21-APX(W41F) dimeric
(Plasmid #58199)

• Purpose: Bacterial expression of APX W41F under control of TetR
• Depositing Lab: Alice Ting
• Publication: Dwyer et al Proc Natl Acad Sci U S A. 2014 May 20;111(20):E2100-9. doi: 10.1073/pnas.1401876111. Epub 2014 May 6.

Backbone

• Vector backbone: pZE
• Backbone size w/o insert (bp): 2226
• Total vector size (bp): 2976
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: pea APX W41F
• Species: Synthetic
• Insert Size (bp): 750
• Mutation: W41F
• Promoter: pL(tetO-1)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: KpnI (not destroyed)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: pLTet-F

Terms and Licenses

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

Depositor Comments

This plasmid encodes the W41F mutant of pea ascorbate peroxidase (APX). The W41F mutation improves enzyme activity. APX W41F is a constitutive homodimer, unlike "APEX", so APX W41F should not be used as a fusion to proteins that are sensitive to dimeric tags.

This plasmid encodes APX W41F under a constitutive promoter, and the promoter is flanked by TetR operator sites. When the plasmid is introduced into cells constitutively expressing the TetR protein, such as the GM1655 Pro cells, then the TetR protein binds to the promoter and suppresses transcription. When a tetracycline drug, such as anhydrotetracycline, is added, the TetR protein is displaced from the promoter, allowing transcription of the APX W41F gene. When the plasmid is introduced into bacterial strains that do not encode a copy of the TetR repressor, expression of APX W41F is constitutive. Any bacterial strain can be used for plasmid propagation.

The pZE plasmid system was developed by Lutz and Bujard, and described in their paper entitled "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" (PMID: 9092630).