2. Samples were taken and cell extracts were separated on a SDS-PAGE gel. Proteins were then transferred to a nitrocellulose membrane, which was probed with antibodies specific for the FLAG peptide (Sigma), ProteinA (Sigma) or GFP (Roche). The membranes were then incubated with HRP-labeled anti-mouse IgG (Sigma), and binding of antibody visualized by scanning with a Syngene Gene Genius Bioimaging System. Affinity isolation of LacI::6 × His A 100 ml culture of strain MG1655lacI::6 × his was grown in LB medium at 37°C to an OD650 of 1.2. Cells were harvested and re-suspended in 4 mls of lysis buffer (10 mM Tris, 100 mM NaCl, 10% Glycerol).

Lysozyme was added to a final concentration FDA-approved Drug Library purchase of 400 μg/ml, and the mixture incubated on ice for MG-132 ic50 30 minutes, with regular mixing. After lysozyme treatment, the lysate was cleared by centrifugation and the supernatant incubated with 200 μl of NTA-Ni-agarose beads (Qiagen), on ice for 30 minutes. The supernatant was then removed, and the beads washed with 1 ml of wash buffer (10 mM Tris, 100 mM NaCl, 10% Glycerol, 10 mM Imidazole). LacI::6 × His was then eluted from the beads with 100 μl of elution buffer

(10 mM Tris, 100 mM NaCl, 10% Glycerol, 250 mM Imidazole). Acknowledgements The Authors would like to thank Prof. C Thomas (University of Birmingham) for the gift of the pEX100T plasmid, and Dr. T Overton (University of Birmingham) for the gift of the pSUB11 plasmid derivative carrying the 3 × FLAG sequence, used in the initial construction of the pDOC-K plasmid. This work was supported by a Wellcome Trust Programme Grant 076689 to SJWB, and BBSRC grant BB/E01044X/1 to CWP, JLH and MJP. The Birmingham Functional Interleukin-2 receptor Genomics laboratory was supported by a Joint Infrastructure Fund grant JIF13209. The strains and plasmids generated in this work are freely available upon request. Electronic supplementary material Additional file 1: Annotated sequence of the pDOC plasmids. The file contains the DNA sequence of each pDOC plasmid with annotation of

open reading frames, multi-cloning sites and primer binding sites. (DOC 218 KB) References 1. Court DL, Sawitzke JA, Thomason LC: Genetic engineering using homologous recombination. Annu Rev Genet 2002, 36:361–388.CrossRefPubMed 2. Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000,97(12):6640–6645.CrossRefPubMed 3. Ellis HM, Yu D, DiTizio T, Court DL: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA 2001,98(12):6742–6746.CrossRefPubMed 4. Herring CD, Glasner JD, Blattner FR: Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 2003, 311:153–163.CrossRefPubMed 5. Murphy KC: Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli.