Next, 1 U of RNasin, 2 μl of 100 mM DTT, 1 μl of 10 mM dNTP and 0.5 μl of 200 U/μl MMLV High Performance Reverse Transcriptase (Epicentre, Madison, WI) were added to each RNA/primer mixture and incubated at 37°C for 1 h, followed by heating at 85°C for 10 min to inactivate the PLX3397 purchase enzyme and then chilled on ice for at least 1 min. The specific cDNA that we prepared was used in the following quantitative real-time
PCR analysis. The components of real-time PCR were prepared by adding 10 ng of each specific cDNA and 1 μl of a 10 mM primer solution to 2 × Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) and adjusted with ddH2O to a final volume of 20 μl. Cycling conditions were performed using Roche LightCycler 2.0 system (Roche Applied selleck chemicals Science, Branford, CT) as follows: 95°C for 2 min followed by 40 cycles of 95°C for 30 sec, 50°C for 30 sec and 72°C for 15 sec. Dissociation curves and non-BEZ235 price template controls were included to detect any primer dimerization or other artifacts. The mRNA transcript levels were obtained by the method described by Livak and Schmittgen [37]. Fusion protein construction A carboxy terminal 6 × histidine-tagged fusion to STM0551 was constructed by amplifying stm0551 with primers stm0551-TOPO-F and stm0551-TOPO-R using genomic DNA of S. Typhimurium LB5010 as the template. The resulting 316-bp PCR
product was cloned into the pET101/D-TOPO vector (Invitrogen, Carlsbad, CA) giving rise to plasmid pSTM0551-His. This recombinant plasmid was sequenced at the adjacent portion of the cloning site to make sure it was in frame before subsequent transformation step. BL21Star™ (DE3) One Shot® chemically competent E. coli (Invitrogen) cells were transformed with pSTM0551-His. Log phase cultures were
induced to express STM0551-His by adding 1 mM IPTG at 37°C for 4 hr. The STM0551-His fusion protein was further purified by ProBond purification kit (Invitrogen) using the protocol provided by the manufacturer. The protein concentration was determined using the Bradford reagent (Fermentas) [38]. A mutant allele of stm0551 was constructed by site-directed mutagenesis using overlapping-extension PCR of S. Typhimurium LB5010 strain genomic DNA Molecular motor template and mutagenic oligonucleotides E49A-TOPO-F and E49A-TOPO-R [39]. Briefly, STM0551-TOPO-F and E49A-TOPO-R were used to amplify the first DNA fragment using Pfu DNA polymerase (Fermentas). The PCR conditions were: denaturing at 94°C for 3 min followed by 35 cycles of 94°C for 45 sec, 50°C for 45 sec and 72°C for 45 sec. The second DNA fragment was amplified using E49A-TOPO-F and STM0551-TOPO-R with the same procedure described above. These two DNA fragments were purified by Montage Gel Extraction Kit (Millipore, Billerica, MA).