Recombination was confirmed by PCR and sequencing, using oligonucleotide primers homologous to chromosomal DNA flanking the modified region (sequencing provided by the Birmingham Functional Genomics laboratory). Note: in GSK1210151A manufacturer addition, dilutions of the culture were routinely plated onto LB agar plates and LB agar plates supplemented with 200 μg/ml of ampicillin, to quantify the amount of donor plasmid digestion by I-SceI and LB agar plates and LB agar plates supplemented with 35 μg/ml chloramphenicol, to quantify pACBSCE digestion by I-SceI. Construction of pDOC derivatives for generating lacI gene fusions Four

different lacI gene fusions {Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|buy Anti-diabetic Compound Library|Anti-diabetic Compound Library ic50|Anti-diabetic Compound Library price|Anti-diabetic Compound Library cost|Anti-diabetic Compound Library solubility dmso|Anti-diabetic Compound Library purchase|Anti-diabetic Compound Library manufacturer|Anti-diabetic Compound Library research buy|Anti-diabetic Compound Library order|Anti-diabetic Compound Library mouse|Anti-diabetic Compound Library chemical structure|Anti-diabetic Compound Library mw|Anti-diabetic Compound Library molecular weight|Anti-diabetic Compound Library datasheet|Anti-diabetic Compound Library supplier|Anti-diabetic Compound Library in vitro|Anti-diabetic Compound Library cell line|Anti-diabetic Compound Library concentration|Anti-diabetic Compound Library nmr|Anti-diabetic Compound Library in vivo|Anti-diabetic Compound Library clinical trial|Anti-diabetic Compound Library cell assay|Anti-diabetic Compound Library screening|Anti-diabetic Compound Library high throughput|buy Antidiabetic Compound Library|Antidiabetic Compound Library ic50|Antidiabetic Compound Library price|Antidiabetic Compound Library cost|Antidiabetic Compound Library solubility dmso|Antidiabetic Compound Library purchase|Antidiabetic Compound Library manufacturer|Antidiabetic Compound Library research buy|Antidiabetic Compound Library order|Antidiabetic Compound Library chemical structure|Antidiabetic Compound Library datasheet|Antidiabetic Compound Library supplier|Antidiabetic Compound Library in vitro|Antidiabetic Compound Library cell line|Antidiabetic Compound Library concentration|Antidiabetic Compound Library clinical trial|Antidiabetic Compound Library cell assay|Antidiabetic Compound Library screening|Antidiabetic Compound Library high throughput|Anti-diabetic Compound high throughput screening| were constructed in MG1655, producing the following recombinant proteins; LacI::6 × His, BIX 1294 datasheet LacI::3 × FLAG, LacI::4 × ProteinA and LacI::GFP. For the LacI::6 × His construct, two primers were designed to amplify the 6 × his coding region and the kanamycin cassette

from pDOC-H: the first primer, D60113, included 27 bp of homology to the C-terminus of lacI, excluding the stop codon, and 18 bp homology to pDOC-H and was designed so that the 6 × his sequence was in frame with the lacI coding sequence. The second primer, D60114 included 27 bp of homology to the region immediately downstream of lacI, and homology to the P-REV annealing sequence. These primers were used to amplify the kanamycin resistance cassette, using pDOC-H as a template, and a proof-reading thermostable DNA polymerase that produces a blunt-ended amplicon. The resulting fragment was blunt end ligated into the EcoRV site of pDOC-C. The cloned region was sequenced using primers D58793 and D58794, which anneal to the S1 and S2 sites (Figure 2) in the pDOC-C plasmid. The resulting plasmid was then used to tag the chromosomal lacI gene in E. coli strain MG1655 by gene doctoring. Recombinants were checked by PCR and sequencing using primers D61347, which anneals within the lacI gene, and D57785, which anneals to the CC1 sequence shown in Figure 2. The lacI::3 × FLAG, lacI::4 × ProteinA and lacI::GFP gene fusions

were made using longer regions of homology to the chromosome, cloned directly into the pDOC-F, pDOC-P and pDOC-G cloning regions. The C-terminal 200 bp of the lacI many gene, excluding the stop codon, was amplified by PCR using primers D59400 and D59401, and cloned into CR1 of the appropriate tagging vector, on a EcoRI:KpnI fragment, arranged so that the coding sequence of the gene was in frame with the epitope tag. Next, a 200 bp region of the lacZ gene (codons 130-205) was amplified by PCR using primers D59402 and D59403 and cloned into CR2 of the appropriate tagging vector, on a XhoI:NheI fragment. The resulting plasmids were then used to tag the chromosomal lacI gene in E. coli strain MG1655 by gene doctoring. Recombinants were checked by PCR and DNA sequencing as before.