PurposeExpression of single-cysteine variant of mature phosphate binding protein, PstS, as scaffold for phosphate biosensor
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||78198||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 (DE3) for protein expression
Copy numberHigh Copy
Gene/Insert namepstS (A197C)
MutationA197C (please see depositors comment below)
- Promoter T7
- Cloning method Restriction Enzyme
- 5′ cloning site NdeI (unknown if destroyed)
- 3′ cloning site XhoI (unknown if destroyed)
- 5′ sequencing primer T7
- 3′ sequencing primer T7 terminal (Common Sequencing Primers)
Please note- The numbering system used was for the mature natural wild-type PstS protein. The plasmid produces an extra N-terminal methionine so all mutations will be off by 1nt (e.g., A197C =A198C)
The expressed protein is a single-cysteine variant of E. coli phosphate binding protein. The gene encodes the mature protein without the N-terminal signal peptide, but with an N-terminal methionine for subsequent labeling at the cysteine with one MDCC (N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide) per molecule. This adduct is then used as a fluorescent phosphate biosensor, having ~7-fold fluorescence intensity increase on binding inorganic phosphate with very high affinity.
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:pET22b_PstS_1 was a gift from Martin Webb (Addgene plasmid # 78198 ; http://n2t.net/addgene:78198 ; RRID:Addgene_78198)
For your References section:Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range. Solscheid C, Kunzelmann S, Davis CT, Hunter JL, Nofer A, Webb MR. Biochemistry. 2015 Aug 18;54(32):5054-62. doi: 10.1021/acs.biochem.5b00449. Epub 2015 Aug 4. 10.1021/acs.biochem.5b00449 PubMed 26199994