The mutagenesis was carried out with QuickChangeII Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). To construct the gene encoding the histidine-tagged MexT, mexT was amplified by PCR using the primers Nde-T1 and Xho-T2 (Table 2). The PCR products were treated with NdeI and XhoI, and inserted into pET-21a(+) (Novagen, Madison, WI) carrying a hexahistidine gene to be attached to the end of the target protein gene, yielding pET21a-MexT-(His)6.
Escherichia coli Origami(DE3)(pLysS) cells were transformed with pET21a-MexT-(His)6. To obtain the MexT-(His)6 recombinant protein, the cells were grown at 37 °C in 500 mL of LB broth, and 0.5 mM IPTG was added. The flask was shaken for an additional 24 h at 22 °C, and the MexT-(His)6 protein was purified from cell-free extracts by chromatography with a column of Profinity IMAC NVP-BKM120 cell line Ni-Charged Resin according to the manufacturer’s instructions (Bio-Rad). The MexT protein purified by this method appeared electrophoretically homogeneous. The 230-bp mexT-mexE intergenic DNA was amplified by PCR using AlexaFluor488-labeled primers pME4510-1Alexa and pME4510-2Alexa (Table 2). The PCR products were isolated from an agarose gel using the QIAquick Gel Extraction kit (Qiagen, GmbH, Hilden, Germany). A 20 μL volume of the reaction mixture containing 230 bp of labeled probe DNA (50 nM) and an appropriate
amount of homogeneously purified MexT-(His)6 was incubated http://www.selleckchem.com/products/Roscovitine.html for 20 min at 24 °C. An aliquot (10 μL) of the mixture was subjected to electrophoresis in 5% polyacrylamide gels (in 0.5 × TBE) at 50 V and 4 °C. The PAK6 results were analyzed with an image analyzer, LAS-4000miniEPUV (Fuji Photo Film Co., Tokyo,
Japan). The transcriptional start-point of the mexEF-oprN operon was determined according to the protocol for a 5′ rapid amplification of cDNA ends (5′ RACE) system (version 2) (Invitrogen, Carlsbad, CA). The total bacterial RNA for 5′ RACE was isolated from stationary phase cells of P. aeruginosa PAO1SC grown in LB broth using the Qiagen RNeasy minikit and RNase-free DNase (Promega), according to the manufacturer’s instructions. A mexE-specific primer (5′-CCGGTGAATTCGTCCCACTCG-3′), purified total RNA, and reverse transcriptase were used for the first-strand cDNA synthesis. A homopolymeric tail was then added to the 3′-end of the cDNA by terminal deoxynucleotidyl transferase (TdT) and dCTP. PCR amplification was performed using poly(C)-tailed cDNA as a template, the abridged anchor primer supplied by the manufacturer, and nested gene-specific primer1 (5′-CGTTCAGCGGTTGTTCGATGAC-3′). The PCR products were amplified again using nested gene-specific primer2 (5′-TGGAATTCCATGCCTTGGGTGGTTTCCG-3′) and the abridged universal amplification primer supplied by the manufacturer.
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