pET22b_PfSSB
(Plasmid #103000)

• Purpose: The expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB
• 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
• Species: Plasmodium falciparum
• 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_WT information
• A portion of this plasmid was derived from a plasmid made by: Synthetically produced by GenScript (The Biology CRO, U.S.A.)
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

The expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB, for subsequent labeling at the single, native 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 ~20-fold fluorescence increase on binding ssDNA with very high affinity. 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.
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.