BW23473菌株pir+,RED重组系统配套菌株-用于R6Kγ复制子质粒扩增(for pKD46, pKD3,pkD4, pKD13, pKD32. etc.)

Description

CGSC Strain#: 7837
Strain Designation: BW23473      Source of Strain: B.L. Wanner
Sex: F- Chromosomal Markers: Δ(argF-lac)169, ΔuidA3::pir⁺, recA1, rpoS396(Am), endA9(del-ins)::FRT, rph-1, hsdR514, rob-1, creC510
Strain Comments:

• Δ(argF-lac)169-- extends from mmuP through orfs preceding argF, through lac to mhpD, literally Δ(mmuP-mhpD)169. (Peters et al. 2003 JB 185:2017)

• Δ(argF-lac)169-- from strain Hfr3000 U169 was initially called ΔlacU169 and described as a lacZY mutation until found to include argF and lacI.

• 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).

• rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

• creC510-- The constitutive phenotype is due to an R77P amino acid substitution

• creC510 was formerly called phoM510

• endA9(del-ins)::FRT was formerly called endA_BT333

• ΔuidA3::pir⁺ was formerly called uidA(Δ MluI)::pir⁺

• Am = amber(UAG) mutation

• Const = constitutive

• del-ins = deletion-insertion

• Haldimann, A., L.L. Daniels, B.L. Wanner 1998. Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J.Bacteriol. 180:1277-1286

• Lu, F., M.A. Schumacher, D.N. Arvidson, A. Haldimann, B.L. Wanner, H. Zalkin, R.G. Brennan 1998. Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190. Biochemistry 37:971-982

• Haldimann, A, BL Wanner 2001. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J. Bacteriol. 183(21):6384-93.

Recovery

1.Obtainan LB agar plate with the appropriate antibiotic.

2.Usinga sterile pipette tip, touch the bacteria growing within the punctured area ofthe stab culture. (A sterilized wire loop or sterile toothpick can be used inplace of a sterile pipette tip.)

3.Runthis tip lightly over a section of the plate, as shown in the figure, to createstreak #1.

4.Usinganother sterile pipette tip, pass through streak #1 and spread the bacteriaover a second section of the plate, to create streak #2.

5.Usinga third sterile pipette tip, pass through streak #2 and spread the bacteriaover the last section of the plate, to create streak #3.

6.Growovernight in a 37 P^oP Cincubator (unless a different growth temperature is indicated on the plasmiddatasheet).

7.Inthe morning, single colonies should be visible. If the bacterial growth is toodense, re-streak onto a new agar plate to obtain single colonies.