pAOJ15
(Plasmid #91567)

• Purpose: (Empty Backbone) Broad host range allelic exchange suicide vector for bacteria - Tse2 toxin driven by anhydrotetracycline inducible promoter
• Depositing Lab: Harry Mobley
• Publication: Armbruster et al PLoS Pathog. 2017 Jun 14;13(6):e1006434. doi: 10.1371/journal.ppat.1006434.

Backbone

• Vector backbone: pRK415
• Backbone manufacturer: N/A
• Backbone size (bp): 10690
• Modifications to backbone: Only the RK2 OriV region, LacZalpha gene, TetR gene and the RP4/RK2 origin of transfer remain from the original plasmid.
• Vector type: Bacterial Expression
• Promoter: Tetracycline promoter

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin+ 1% Arabinose
• Growth Temperature: 30°C
• Growth Strain(s): E. coli EPI300
• Growth instructions: Requires media containing 1% arabinose for plasmid replication, as pAOJ15 needs the TrfA protein supplied in trans. EPI300 expresses TrfA under control of the arabinose promoter. Growth at 30° is not strictly necessary, as the plasmid is NOT temperature sensitive, but this temperature seems to result in better miniprep yields in my hands.
• Copy number: Unknown

Cloning Information

• Cloning method: Gibson Cloning

Resource Information

• Supplemental Documents:
  • pAOJ15.gbk
  • pAOJ15 seqbuilder file

Terms and Licenses

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

Depositor Comments

Please note- 2 mutations in tse2 (H30R and K33Q) were identified during Addgene's quality control process. The depositing lab has noted that these mutations have no affect on plasmid function, and are likely a strain variant.

The Pseudomonas aeruginosa PAO1 Tse2 toxin on the backbone acts as a counter selection marker for allelic exchange. Expression of Tse2 is highly toxic to a huge variety of cells, and can result in less background than traditional markers like SacB.
See: Khetrapal, Varnica et al. “A Set of Powerful Negative Selection Systems for Unmodified Enterobacteriaceae.” Nucleic Acids Research 43.13 (2015): e83. PMC. Web. 11 Apr. 2017.

Toxin expression is tightly repressed by TetR, until induction by anhydrotetracycline. Anhydrotetracycline is non-toxic to most bacteria, and freely crosses membrane barriers without need of specific transporters, potentially enabling this vector to work in a wide variety of bacteria.

The ampicillin resistance gene is flanked by two BsaI sites. It can be cleanly removed and replaced with another marker via Golden Gate cloning.

If to be used for mating, this plasmid or derivatives can be transformed into commonly used strains such as S17-1λpir, SM10, DH5a(pRK2013), because these strains provide the RK2/RP4 TrfA protein in trans.

Important: This suicide plasmid IS NOT based on the R6K gamma ori like most suicide plasmids, so it won't replicate in strains like DH5aλpir.