pET22b_PfSSB_W166C
(Plasmid #103001)

• Purpose: The expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB (C93A,W166C), used for obtaining a fluorescent adduct at the cysteine
• Depositing Lab: Martin Webb
• Publication: Chisty et al PLoS One. 2018 Feb 21;13(2):e0193272. doi: 10.1371/journal.pone.0193272. eCollection 2018.

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: BL21-DE3 pLysS for protein production
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: PfSSB_W166C
• Species: Plasmodium falciparum
• Mutation: C93A, W166C
• Promoter: T7

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (unknown if destroyed)
• 3′ cloning site: XhoI (unknown if destroyed)
• 5′ sequencing primer: T7

Resource Information

• Supplemental Documents:
  • pET22b_PfSSB_W166C information
• A portion of this plasmid was derived from a plasmid made by: Synthetically produced by GenScript (The Biology CRO, U.S.A.)

Terms and Licenses

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

Depositor Comments

Usage:
The expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB, with two point mutations, C93A and W166C, for subsequent labeling at the 166 cysteine with one IDCC (N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide) or Cy3B per subunit. PfSSB was expressed without the N-terminal apicoplast localisation sequence (76 amino acids) but amino acids are numbered for the full-length protein, that is the first amino acid in the expressed protein is M77.W166C is the equivalent position to W88 in E. coli SSB, which was labeled with Cy3B for single molecule measurements of DNA unwinding using TIRF (Fili, N.; Mashanov, G.; Toseland, C. P.; Batters, C.; Wallace, M. I.; Yeeles, J. T. P.; Dillingham, M. S.; Webb, M. R.; Molloy, J. E., Visualizing DNA unwinding by helicases at the single molecule level. Nucleic Acids Res. 2010, 38, 4448-4457.)
Protocol:
See Hedgethorne and Webb (2012), which is also followed for PfSSB purification, except with the omission of the PEI precipitation step.
Hedgethorne, K.; Webb, M. R., Fluorescent SSB as a reagentless biosensor for single-stranded DNA. Methods Mol. Biol. 2012, 922, 219-233.