pET22b_EcSSB_1
(Plasmid #78203)

• Purpose: Expression of single-cysteine variant of single-stranded DNA-binding protein protein, EcSSB, as scaffold for ssDNA biosensor
• Depositing Lab: Martin Webb
• Publication: Dillingham et al Biophys J. 2008 Oct;95(7):3330-9. doi: 10.1529/biophysj.108.133512. Epub 2008 Jul 3.

Backbone

• Vector backbone: pET22b
• 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-DE3 pLysS for protein expression
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ssb (G26C)
• Species: E. coli
• Mutation: G26C (please see depositor comment below)
• Promoter: T7

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 terminal

Resource Information

• Supplemental Documents:
  • pET22b_EcSSB_1 information

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.