PurposeExpression of single-cysteine variant of plasmid segregation protein, ParM, as scaffold for ADP biosensor
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||78201||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
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 I27C T174A T175N C287A)
MutationK33A I27C T174A T175N 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, 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.
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 _1 was a gift from Martin Webb (Addgene plasmid # 78201 ; http://n2t.net/addgene:78201 ; RRID:Addgene_78201)
For your References section:A biosensor for fluorescent determination of ADP with high time resolution. Kunzelmann S, Webb MR. J Biol Chem. 2009 Nov 27;284(48):33130-8. doi: 10.1074/jbc.M109.047118. Epub 2009 Sep 29. 10.1074/jbc.M109.047118 PubMed 19801632