PurposeExpression of single-cysteine variant of single-stranded DNA-binding protein protein, EcSSB, as scaffold for ssDNA biosensor
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||78203||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-DE3 pLysS for protein expression
Copy numberHigh Copy
Gene/Insert namessb (G26C)
MutationG26C (please see depositor comment below)
- Promoter T7
- 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 terminal (Common Sequencing Primers)
Please note: The numbering system used was for the mature natural wild-type ssb protein. The N-terminal methionine is quantitatively cleaved off in E. coli and was not numbered so all mutations will be off by 1 nucleotide (e.g., G27C = G26C).
The expressed protein is a single-cysteine variant of E. coli single-stranded DNA-binding protein, EcSSB, for subsequent labeling at the cysteine with one IDCC (N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide) per subunit. This adduct is then used as a fluorescent single-stranded DNA (ssDNA) biosensor, having ~6-fold fluorescence increase on binding ssDNA 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_EcSSB_1 was a gift from Martin Webb (Addgene plasmid # 78203 ; http://n2t.net/addgene:78203 ; RRID:Addgene_78203)
For your References section:Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity. Dillingham MS, Tibbles KL, Hunter JL, Bell JC, Kowalczykowski SC, Webb MR. Biophys J. 2008 Oct;95(7):3330-9. doi: 10.1529/biophysj.108.133512. Epub 2008 Jul 3. 10.1529/biophysj.108.133512 PubMed 18599625