CGSC Strain#: 12551
Strain Designation: BW20767/pRL27      Source of Strain: W.W. Metcalf
Sex: F- Episome/Plasmid: pRL27
   Plasmid Markers/Mutations: oriT_RP4, kan, oriR6K_γ, tetAp-tnp
   Plasmid Comment: pRL27 is a low-copy pir-dependent conjugative plasmid that encodes the kanamycin resistant hyperactive Tn5 system of Reznicoff.
Chromosomal Markers: RP4-2(Km::Tn7,Tc::Mu-1), leu-163::IS10, ΔuidA3::pir⁺, recA1, endA1, thiE1, hsdR17, creC510
Strain Comments:

• This plasmid is known to be somewhat unstable, presumably because of constitutive expression of the of the transposase. As a result mutations in the tnp gene tend to accumulate and these mutants will not work for mutagenesis.
• It is suggested that you immediately test several colonies to verify their ability transfer the transposon. Once this is verified, freeze the strain immediately and minimize the passage of the strain to reduce the accumulation of tnp mutants.
• RP4-2(Km::Tn7,Tc::Mu-1)-- integrated plasmid is Kan and Tet sensitive, due to the insertions in Tc and Km, and the Tn7 results in TRIM/aminopterin resistance and low level strR.
• RP4-2(Km::Tn7,Tc::Mu-1)-- The RP4-2 also has an ISR1 insertion element within the Tn1³⁵ transposon that inactivates the adjacent ampicillin resistance gene.
• leu-163::IS10-- This allele was created by selecting for a spontaneous TetS version of leu-63::Tn10
• ΔuidA3::pir⁺-- Allows the replication of plasmids with an R6Kγ origin of replication
• recA1-- : Missense mutation, altered isoelectric point. Sequenced: G to A for Nuc. 720 (Gly 160 Asp).
• endA1-- G to A transition mutation resulting in E208K amino acid substitution.
• creC510-- The constitutive phenotype is due to an R77P amino acid substitution
• creC510 was formerly called phoM510
• thiE1 was formerly called thi-1
• ΔuidA3::pir⁺ was formerly called uidA(Δ MluI)::pir⁺
• = good miniprep dsDNA quality
• Const = constitutive
• = kanamycin resistant

References:

• Metcalf, WW, W Jiang, LL Daniels, SK Kim, A Haldimann, BL Wanner 1996. Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid 35(1):1-13.
• Larsen, RA, MM Wilson, AM Guss, WW Metcalf 2002. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178(3):193-201.