Overnight 30°C cultures of 1572lux and 1668lux were diluted 1:200 in fresh LB with antibiotics and grown for 3-4 h at 30°C. The optical density of the culture was adjusted to a starting OD600 of 0.1 and 200 μl was added

to the wells of the 96 well plate. Assays were performed at 24°C, 28°C and 37°C. Luminescence and OD at 405 nm of the cultures was automatically determined every 30 min for 18 h and presented as relative light units per unit of OD405 (light per unit cell). For every promoter PI3K Inhibitor Library in vivo fusion assay each sample was assayed in triplicate on the 96-well plate, and each experiment was carried out in duplicate. Construction selleck of Maltose Binding Protein (MBP) fusion proteins Primers YptbIntMBP-1 and YptbIntMBP-2 (Table 2) were used

to PCR amplify the 3′ end (1044 bp) of the ifp coding sequence. This PCR product was cloned into the pMAL-p2x vector (NEB, Hitchin, UK) using EcoRI and XbaI enzyme restriction sites which had been incorporated see more into the primer design. In order to generate an MBP-Ifp fusion with the terminal cysteine (Cys1070) mutated to a glycine (MBP-IfpC337G), primer YptbIntMBP-3 was substituted for YptbMBP-2. Primer YptbIntMBP-3 contained an alternative sequence (underlined in table 2) to mutate the terminal cysteine to a glycine. MBP-fusion proteins were then expressed in TB1 E. coli. Purification of MBP-Ifp and MBP-IfpC337G E. coli transformed with the MBP-fusion plasmids were cultured for 2 h at 37°C using 5 ml of an overnight culture in 250 ml LB broth with 2 mM glucose and ampicillin until a culture growth of OD600 0.5 was reached. Expression of the fusion protein was induced with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) then the culture was incubated for further 4��8C 2 hours at 37°C. The cultures were centrifuged and pellets stored overnight at -20°C then resuspended

in column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA in H2O). The cells were lysed by sonication using a Bioruptor sonicator (Diagenode; 60 second pulses with a 30 second recovery period). Insoluble proteins were removed by centrifugation and the supernatant was applied to 1 ml columns of amylose resin (NEB). After washing with 15 ml column buffer, proteins were eluted with 10 ml column buffer/10 mM maltose. Proteins were concentrated using Amicon ultra 50 kDa columns (Millipore, Watford, UK), and then confirmed by Coomassie staining and western blotting with anti-MBP (NEB). Protein concentrations were determined by Pierce BCA protein assay kit (Rockford, USA) according to the manufacturer’s protocol. Analysis of MBP-fusion protein binding to HEp-2 cells by fluorescence microscopy Binding of MBP-tagged Ifp was determined by fluorescence microscopy as described previously [18]. HEp-2 cells were cultured overnight on glass coverslips in 24-well plates at 2.