pET22b_EcSSB_2
(Plasmid #78204)

• Purpose: Expression of single-cysteine variant of single-stranded DNA-binding protein protein, EcSSB, as scaffold for ssDNA biosensor
• Depositing Lab: Martin Webb
• Publication: Fili et al Nucleic Acids Res. 2010 Jul;38(13):4448-57. doi: 10.1093/nar/gkq173. Epub 2010 Mar 28.

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 (W88C)
• Species: E. coli
• Mutation: W88C (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_2 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., W89C = W88C).

The expressed protein is a single-cysteine variant of E. coli single-stranded DNA-binding protein, EcSSB, for subsequent labeling at the cysteine. The adduct with Cy3B maleimide has been used as a biosensor for single-stranded DNA in TIRF microscopy measurements.