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发布于:2019-5-27 17:42:13  访问:68 次 回复:0 篇
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(unrooted). The tree was inferred with IQ-TREE along with the LG+4 model.
For cloning into pILL2157, the genes had been PCR-amplified in the B128 chromosome employing the couple of primers pil-UP/pil-DO and cloned into the NdeI/EcoRV web-sites (for niuD and niuDE) or the NdeI/BamHI web pages (for niuB1 and niuB2) of pILL2157.[74] and also the supplier (Fermentas) 3-MethylindoleFormula suggestions. pylori mutants were obtained by organic transformation of B128-S, a streptomycin resistant derivative of B128, with 1 g of a preparation with the suicide plasmid DNA and choice on blood agar plates containing chroramphenicol 6 g.mL-1 (for G27 and B128-S) or 10 g.mL-1 (for SS1) as described previously [75].[74] and the supplier (Fermentas) recommendations. NucleoBond Xtra Midi Kit (Macherey-Nagel) and QIAamp DNA Mini Kit (Qiagen) were employed for plasmid preparations and H. pylori genomic DNA extractions, respectively.[74] and also the supplier (Fermentas) suggestions. NucleoBond Xtra Midi Kit (Macherey-Nagel) and QIAamp DNA Mini Kit (Qiagen) have been utilized for plasmid preparations and H. pylori genomic DNA extractions, respectively. PCR have been performed either with Taq Core DNA polymerase (MP Biomedicals), or with Phusion Hot Start off DNA polymerase (Finnzymes) when the item expected high fidelity polymerase. The pGEMT vector (Promega) was used to construct in E. coli the suicide plasmids that served for mutagenesis in H. pylori.Construction of unmarked H. pylori mutantsUnmarked niuD and niuB deletion mutants of H. pylori were constructed by allelic exchange as described [53] applying H. pylori suicide plasmids derived from pGEMT, in which about 500 bp with the 5‘-end plus the 3‘-end regions quickly flanking the open reading frame with the target gene have been cloned using PCR fragments generated with the primers indicated in S2 Table on each side of a difH-cat-rpsL-difH cassette amplified with primers difHrpsLcat-1 and difHrpsLcat-2. The H. pylori mutants were obtained by all-natural transformation of B128-S, a streptomycin resistant derivative of B128, with 1 g of a preparation with the suicide plasmid DNA and choice on blood agar plates containing chroramphenicol 6 g.mL-1 (for G27 and B128-S) or 10 g.mL-1 (for SS1) as described previously [75]. Removal of the cassette was achieved by plating the CmR clones on blood agar plates containing streptomycin 10 g. mL-1. Deletion of the gene of interest was verified by PCR and sequencing in the gene area.Building of plasmids and complemented mutantsThe mutants were complemented by way of two approaches. One was by introducing the wild variety copy on the gene to be complemented beneath the control of your ureI promoter around the chromosome, at a neutral web site that was shown to influence neither in vitro growth nor mouse colonization as described in [22,54]. The corresponding constructs were designated "c-X" for chromosomally inserted, X becoming the name in the gene. The second one particular was by cloning the gene under the handle of an IPTG inducible promoter PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22928863 within the pILL2157 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/19808328 E. coli/H. pylori shuttle vector [76], the corresponding constructs had been designated "p-X" for inserted on a plasmid, X getting the name of the gene.
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