pHis17_ParM _1
(Plasmid #78201)

• Purpose: Expression of single-cysteine variant of plasmid segregation protein, ParM, as scaffold for ADP biosensor
• Depositing Lab: Martin Webb
• Publication: Kunzelmann et al J Biol Chem. 2009 Nov 27;284(48):33130-8. doi: 10.1074/jbc.M109.047118. Epub 2009 Sep 29.

Backbone

• Vector backbone: pHis17
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: use BL21 Ai for protein expression.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: parM (K33A I27C T174A T175N C287A)
• Species: E. coli
• Mutation: K33A I27C T174A T175N C287A
• Promoter: unknown
• Tag / Fusion Protein:
  • His6 (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Unknown (unknown if destroyed)
• 3′ cloning site: Unknown (unknown if destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pHis17_ParM_1 information
  • pHis17_ParM _1 protocol
• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Depositor Comments

The plasmid is used to express the plasmid segregation protein, ParM, from E. col. The protein is modified for use as the scaffold of an ADP biosensor, MDCC-ParM. A single cysteine mutation is for label attachment (I27C); there is a mutation to inhibit filament formation (K33A), two mutations to weaken ATP binding (T174A and T175N), a mutation to change the exposed cysteine (C287A) and a C-terminal His6 tag to aid purification. When labelled with a single N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC) fluorophore, this biosensor has a fluorescence intensity change of up to 4-fold and can be used to measure ADP in the range of tens of nanomolar to tens of micromolar in the presence of up to~200 μM ATP.