pSAM_Ec
(Plasmid #102939)

• Purpose: Transposon mutagenesis and Tn-seq
• Depositing Lab: Matthew Mulvey
• Publication: Wiles et al PLoS Genet. 2013;9(8):e1003716. doi: 10.1371/journal.pgen.1003716. Epub 2013 Aug 22.

Backbone

• Vector backbone: pSAM
• Total vector size (bp): 4559
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin and Kanamycin, 100 & 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): pir+ E. coli strain EcS17
• Growth instructions: EcS17/pSAM-Ec need ~36-48 hours to reach an OD600 of 1.0.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: mariner transposon flanked by MmeI modified inverted repeats and the himar1C9 transposase

Resource Information

• Supplemental Documents:
  • TnSeq_Protocol_pSAM_Ec
• A portion of this plasmid was derived from a plasmid made by: pSAM_Bt: Goodman AL, McNulty NP, Zhao Y, Leip D, Mitra RD, Lozupone CA, Knight R, Gordon JI (2009). Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe, 6: 279–289.
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

The Plac promoter from pGFP-Mut3.1 (Clonetech), along with its associated ribosome binding sequence, was amplified via PCR. Engineered 59 BamHI and 39 NdeI restriction sites were used to sub-clone the resulting fragment into a BamHI/NdeI (New England Biolabs) double digested pSAM_Bt vector upstream of the himar1C9 transposase gene. The kanamycin resistance gene from pKD4 was amplified and ligated using the restriction sites MfeI and XbaI, replacing the erythromycin resistance gene ermG in pSAM_Bt. The resulting transposon mutagenesis vector, pSAM-Ec, was stored and propagated in the pir+ E. coli strain EcS17.