5. Aliquots of 0. 5 ml of every strain containing the clone or even the empty vector had been assayed for B galactosidase action in accordance to Miller, The data had been analyzed working with the program Graph Pad Prism V5. 01. Web site directed mutagenesis A achievable transcription terminator involving dksA and gluQ rs was recognized employing the plan Mfold, Webpage directed mutagenesis by overlap PCR was performed to disrupt the predicted terminator, Using the fragment VCPDT cloned during the vector pTZ57R T as template, was amplified a one,072 bp fragment, which contain the mutation, applying the primers PdksAF and TERMGQ3, when a second fragment of 162 bp overlapping the mutated area, was obtained with primers TERGQ2 and M13R, The two fragments had been digested with DpnI, purified and mixed at equimolar quantities to perform a PCR reaction making use of the 50 and thirty ends primers, The 1,110 bp amplified fragment was cloned within the vector pTZ57R T and sequenced to confirm the mutation.
This plasmid was digested with BamHI and HindIII and also the fragment subcloned in on the vector pQF50. Determination of initial methionine of GluQ RS So as to create that’s the initial AUG codon of gluQ rs, the recombinant plasmid pATGGQRS the full report was constructed. A PCR reaction was carried out applying the primers ATGGQRSF and ATGGQRSR and genomic DNA from S. flexneri. The amplified fragment, containing the BamHI website, prevent codon of dksA, the intergenic region with the terminator, the gluQ rs read ing frame without having its end codon plus the XhoI site was cloned into pET15c, a modified version of pET15b, which was constructed by inserting the 290 bp XbaI and BlpI fragment of pET20b containing the polylinker into pET15b.
This construct allowed the synthesis of the C terminal histidine tagged protein below the transcription management from the T7 promoter. The construct was trans formed in BL21 strain as well as His tagged protein was TW37 partially purified by affinity chromatography as described previously, The eluted protein was trans ferred to a PVDF membrane and stained with Coomassie blue. The predominant band within the expected dimension was sequenced in the Protein Core Facility within the Institute for Cellular and Molecular Biology, University of Texas at Austin. Development of your plasmid for complementation of your gluQ rs mutation This plasmid was constructed through the pATGGQRS plasmid during which the T7 promoter was removed by digestion with BglII and NcoI enzymes and replaced from the TRC promoter obtained from pTRC99a plasmid by amplification and digestion with BamHI and NcoI to obtain the pTRCGQ plasmid.
The empty plasmid was constructed by incorporating the TRC promoter to the pET15c plasmid. Inactivation of gluQ rs gene in S. flexneri Deletion of gluQ rs was carried out employing the red recombinase process together with the following modifica tions.