pSW031
(Plasmid #231144)

• Purpose: Used to express cyclic sortase cW11 (recognizes APXTG motif) from E. coli.
• Depositing Lab: Philip Cole
• Publication: Whedon et al J Am Chem Soc. 2024 Nov 25. doi: 10.1021/jacs.4c12585.

Backbone

• Vector backbone: pTXB1
• Backbone size w/o insert (bp): 5336
• Total vector size (bp): 6248
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Protein expressed from LOBSTR Rosetta(DE3) E. coli. Induced with 0.5 mM IPTG for 3 hrs at 37 degrees C after reaching an OD600 of 0.6-0.8. Overexpression below 37 degrees C results in less enzyme cyclization through intramolecular intein splicing, and more oligomerization through intermolecular intein splicing. The oligomeric form is significantly less active than the cyclic form.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: S. aureus Sortase A mutant
• Alt name: cW11
• Species: S. aureus, N. punctiform
• Insert Size (bp): 912
• Mutation: P94S, E108Q, S116V, D160N, T164Q, V168M, L169H, D170L, E171A, Q172E, K190E, K196T
• Promoter: T7
• Tags / Fusion Proteins:
  • Nostoc punctiform split intein C-terminal fragment (N terminal on insert)
  • Nostoc punctiform split intein N-terminal fragment (C terminal on insert)

Cloning Information

• Cloning method: Other
• 5′ sequencing primer: T7

Resource Information

• Supplemental Documents:
  • pSW031a_cW11_AmpR.gbk
• A portion of this plasmid was derived from a plasmid made by: The gene for F40 sortase was obtained from Dr. Dirk Schwarzer (University of Tübingen), and the gene for the Nostoc punctiform split sortase were obtained from Dr. Champak Chatterjee (University of Washington).

Terms and Licenses

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