pHis17_ParM _2
(Plasmid #78202)

• Purpose: Expression of double-cysteine variant of plasmid segregation protein, ParM, as scaffold for ADP biosensor
• Depositing Lab: Martin Webb
• Publication: Kunzelmann et al ACS Chem Biol. 2010 Apr 16;5(4):415-25. doi: 10.1021/cb9003173.

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 D63C T174A T175N D224C C287A)
• Species: E. coli
• Mutation: K33A D63C T174A T175N D224C 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 _2 information
  • pHis17_ParM _2 protocol
• Articles Citing this Plasmid:
  • 2 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, ATR-ParM. Two cysteine mutations are for label attachment (D63C and D224C); 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 two iodoacetamidotetramethylrhodamine (IATR) fluorophores, this biosensor has a fluorescence intensity change of up to 20-fold and can be used to measure ADP in the range of hundreds of nanomolar to ~100 μM, in the presence of up to millimolar ATP. The biosensor can also be used for measurement of other nucleoside diphosphates e.g. GDP, UDP and others.