This promoter fragment contains the IS5 that increases flhD expression and is located at −1,294 bp to −94 bp [47], making the fragment 1,921 bp in length. The forward and reverse primers were designed with XhoI and BamHI restriction enzyme recognition sites at the Y-27632 5′ ends. The flhD promoter fragment was then digested with XhoI and BamHI. The vector pUA66 (Open Biosystems, Huntsville, AL), containing gfpmut2 as a reporter gene and a kanamycin resistance

cassette, was also digested with these enzymes. To reduce re-ligation of the plasmid, digested pUA66 vector was treated with Calf Intestinal Alkaline Phosphatase (CIAP, Promega, Madison WI) that removes the 5′ phosphate. The double digested flhD promoter region was ligated into the digested and CIAP-treated pUA66 vector. Competent JM109 cells (Promega, Madison WI) were transformed with the resulting plasmid pPS71. The insertion was confirmed by restriction digest and sequencing. Ultimately, pPS71 was transformed into chemically competent AJW678 and AJW2050. pKK12 The antibiotic resistance of pPS71 was changed from KmR to CmR creating pKK12. This Immunology inhibitor permitted transformation of the flhD::gfp fusion plasmid into KmR mutants. pPS71 was digested with EagI to remove 280 bp from pPS71. This deleted region started upstream of the flhD

promoter and extended upstream into the kanamycin resistance gene. This caused Dasatinib research buy inactivation of kanamycin resistance. The digested plasmid was blunt ended with Klenow (Promega, Madison WI), and treated with CIAP. pHP45Ω-Cm was the Carbohydrate source of the chloramphenical resistance gene

cassette [63] and was digested with EcoRI and blunt ended with Klenow. The CIAP-treated pPS71 and pHP45Ω-Cm DNA fragments were ligated. Competent JM109 were transformed with the resulting plasmid pKK12, transformants were resistant to chloramphenicol, but not to kanamycin. Competent AJW2143 (rcsB::Kn) were then transformed with pKK12. pEC2 To construct this plasmid, the rcsB promoter region that starts 100 bp upstream of its +1 transcriptional start site and ends 50 bp downstream was PCR-amplified from AJW678, using 5′-GAGAGATCTGCAACCTGTATCACACCCGATGAAAG-3′ as forward primer and 5′-GCAAAGCTTCGGATGGTCATCGGCAATAATTACG-3′ as reverse primer. The PCR-amplified region was then cleaned up and ligated into pGEM-T Easy (Promega, Madison WI). Successful ligations were identified by white color of the transformed colonies. Plasmids were digested using the HindIII and BglII restriction sites that had been added to the 5′ends of the primers. The promoterless pAcGFP1-1 encodes the green fluorescent protein AcGFP1, a derivative of AcGFP from Aequorea coerulescens, and has a kanamycin resistance gene (Clontech, Mountain View, CA). This plasmid was also double digested with the same enzymes. The digested rcsB promoter region was ligated into the digested pAcGFP1-1 vector.