pZJD11 Genr, pRK2 AZD9291 nmr derived plasmid, lacZ [12] A ppr-strep tag II fusion gene was constructed as follows.
pET16b containing the entire ppr gene (pNB10), as well as pET16b-Pph were cut by NcoI and the resulting fragments (~6.0 kb and ~2.5 kb) were ligated. The orientation of the ppr-insert was checked by DNA-sequencing and the resulting plasmid was named pET16b-Ppr. To construct an arabinose inducible full length ppr, the gene was excised by XbaI and HindIII from pET16b-Ppr and ligated into the pBAD18 vector. The putative phosphorylation site (the histidine at position 670 in the Ppr protein) was changed to an alanine (CAC→GCG) using site directed mutagenesis with the primers (5′-CTGGCGAACATGAGCGCGGAGCTGCGGACTCCG-3′) and (5′-CGGAGTCCGCAGCTCCGCGCTCATGTTCGCCAG-3′) https://www.selleckchem.com/products/MLN-2238.html and pSK4 as a template. The resulting mutant Wnt inhibitor was digested by NdeI and BamHI and subcloned into the pET16b vector generating pET16b-PphH670A. Then the pphH670A mutant was excised by XbaI and HindIII and the fragment
was inserted into the pBAD18 vector to create pBAD-PphH670A. To express the histidine kinase domain Pph with an N-terminal his10-tag and a C-terminal strep-tag II in R. centenaria, the plasmid pZJD11 (kindly provided by C. Bauer) was used [12]. We used the oxygen regulated puc promoter and the puhA Shine Dalgarno sequence from Rhodobacter capsulatus to initiate translation. Therefore, a PCR reaction with the primers (5′-TACGTAGGGCCCTAAGCTAAAGGAGGACTAACATGGGCCATCATCAT-3′)
and (5′-TACGTAGGCGCGAATTCGGCTTGATCAGGC-3′) and pET16b-Pph as a template was conducted. P-type ATPase Simultaneously, a SnaBI restriction site was introduced at the 3′ end of the gene. The resulting fragment was subcloned into pGEM T-easy vector (Promega) and verified by DNA sequencing. This plasmid was used as a template to insert the puc promoter via a second PCR. The primers (5′-GGTAACCTTGATCGCCGACACTTGGGCTCCCA TAGTGGAGCTCGGGCCCTAAG-3′) and (5′-TACGTAGGCGCGAATTCGGCTTGATCA GGC-3′) were used to introduce a BstEII site at the 5′ end. The resulting fragment was inserted into pGEM T-easy vector. After sequencing, the pph construct was excised by BstEII and SnaBI and ligated into the corresponding sites of pZJD11 to generate pSK10. To express the Rc-CheW protein in E. coli, the cheW gene was amplified by PCR from the R. centenaria genome using the primers (5′CATATGCATGCCCGCCTGCCCGTTCCC-3′) and (5′GGGAATCGTTCATTGCGATCAGTTTCCGG-3′), respectively. The resulting fragment was first cloned into pT-Adv.
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