pGC4593
(Plasmid #110206)

• Purpose: Accessory plasmid with inducible TEV protease and unnatural tRNA system (AcF)
• Depositing Labs: Adam Arkin, Guillaume Cambray
• Publication: Cambray et al Nat Biotechnol. 2018 Nov;36(10):1005-1015. doi: 10.1038/nbt.4238. Epub 2018 Sep 24.

Backbone

• Vector backbone: pBROBE
• Modifications to backbone: We modified pEVOLPROBE-aaRS (a gift from Chang Liu, University of California, Irvine) which comprises a pBBR1 origin of replication and kanamycin resistance cassette derived from pBROBE18 and an orthogonal tRNA/aminoacyl-tRNA synthetase system derived from pEVOL19. pEVOLPROBE-aaRS permits to express an optimized tRNACUA and two copies of a cognate aminoacyl-tRNA synthetase (aaRS) gene evolved to selectively discriminate the unnatural amino-acid p-acetylphenylalanine (pAcF). One of the aaRS copy is controlled by a pBAD promoter, which is repressed by the product of the araC gene, also encoded on the plasmid. Upon addition of 0.1% arabinose (Sigma) and 10 mM pAcF (Synchem), tRNACUA are effectively charged with pAcF and outcompete RF1 for the decoding of the amber stop codon (UAG), resulting in the incorporation of the unnatural amino acid to the polypeptide chain and continuation of elongation19. We amplified a chloramphenicol resistance cassette from pBbE1c-RFP20 and cloned it in place of the original kanamycin resistance cassette (NotI/XbaI) to yield plasmid pGC4510. We amplified the tev protease gene from pRK60321 and cloned it into pBbA2k (EcoRI/BamHI)20, yielding pGC2109. A fragment containing anti-oriented tev and tetR genes driven by a bidirectional pTet promoter was amplified from pGC2109 and cloned in pGC4510 (NotI), to yield the accessory plasmid pGC4593. Addition of 200 nM anhydrotetracycline permits the expression of the TEV protease, which recognize and cuts a specific polypeptide motif22.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: TEV protease
• Promoter: Ptet

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: NA
• 3′ sequencing primer: NA

Gene/Insert 2

• Gene/Insert name: aaRS
• Promoter: Pbad

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: NA
• 3′ sequencing primer: NA

Resource Information

• Supplemental Documents:
  • pGC4593.gb
  • cconstruction description
• A portion of this plasmid was derived from a plasmid made by: Chang Liu, UC Irvine

Terms and Licenses

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