PurposeExpression of double-cysteine variant of plasmid segregation protein, ParM, as scaffold for ADP biosensor
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||78202||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Vector typeBacterial Expression
Growth in Bacteria
Growth instructionsuse BL21 Ai for protein expression
Copy numberHigh Copy
Gene/Insert nameparM (K33A D63C T174A T175N D224C C287A)
MutationK33A D63C T174A T175N D224C C287A
- Promoter unknown
/ Fusion Protein
- His6 (C terminal on backbone)
- 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 (Common Sequencing Primers)
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pHis17_ParM _2 was a gift from Martin Webb (Addgene plasmid # 78202 ; http://n2t.net/addgene:78202 ; RRID:Addgene_78202)
For your References section:A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM. Kunzelmann S, Webb MR. ACS Chem Biol. 2010 Apr 16;5(4):415-25. doi: 10.1021/cb9003173. 10.1021/cb9003173 PubMed 20158267