pIN206
(Plasmid #255797)

• Purpose: Expression of the xenotetrapeptide synthetase NRPS from three fragments using split inteins gp41-1 and SspGyrB in E. coli.
• Depositing Lab: Helge Bode
• Publication: Split inteins for generating combinatorial non-ribosomal peptide libraries (unpublished)

Backbone

• Vector backbone: pCDF
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Spectinomycin and Streptomycin, 50 & 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: Use E. coli strain DH10B::mtaA (Addgene #255799) for NRPS protein expression.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: SspGyrBC - XtpS
• Species: Xenorhabdus nematophila ATCC 19061
• Tag / Fusion Protein:
  • His6-tag (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning

Terms and Licenses

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

Depositor Comments

This plasmid encodes the termination unit of the non-ribosomal peptide synthetase (NRPS) called xenotetrapeptide synthetase (XtpS), split at an XUC site and fused to the C-terminal part of the split intein SspGyrB.When co-expressed with pIN097 and pIN202, the three fragments splice into a functional NRPS that produces the non-ribosomal peptide (NRP) called xenotetrapeptide (XTP; cyclo[Leu-D-Val-Val-D-Val]; CC(C)C[C@H]1C(=O)N[C@@H](C(=O)N[C@H](C(=O)N[C@@H](C(=O)N1)C(C)C)C(C)C)C(C)C; 410.6 g/mol).

For successful NRP production, the three plasmids must be co-transformed into an E. coli strain that expresses a broad-spectrum phosphopantetheinyl transferase, such as MtaA (DH10B::mtaA) or Sfp (BAP1), which is required for the post-translational activation of the T domains.

Expression is controlled by an araBAD promoter, inducible with 0.2% arabinose.