pET22b_PfSSB_W166C
(Plasmid
#103001)
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PurposeThe expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB (C93A,W166C), used for obtaining a fluorescent adduct at the cysteine
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 103001 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepET22B
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Growth instructionsBL21-DE3 pLysS for protein production
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert namePfSSB_W166C
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SpeciesPlasmodium falciparum
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MutationC93A, 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 (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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A portion of this plasmid was derived from a plasmid made bySynthetically produced by GenScript (The Biology CRO, U.S.A.)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
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.
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.
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For your Materials & Methods section:
pET22b_PfSSB_W166C was a gift from Martin Webb (Addgene plasmid # 103001 ; http://n2t.net/addgene:103001 ; RRID:Addgene_103001) -
For your References section:
Fluorescent single-stranded DNA-binding protein from Plasmodium falciparum as a biosensor for single-stranded DNA. Chisty LT, Quaglia D, Webb MR. PLoS One. 2018 Feb 21;13(2):e0193272. doi: 10.1371/journal.pone.0193272. eCollection 2018. PONE-D-17-36543 [pii] PubMed 29466468