Firstly, a multiple cloning site (MCS) was introduced into the commercially available, mobilisable broad host range vector pBHR1 (MoBiTec; KmR, CmR) by excising a 1.6-kb BstB I fragment containing a MCS within the lacZ gene and the chloramphenicol resistance gene from plasmid pBBR-MCS1  and cloning
it into the 4.5-kb fragment that resulted from cutting pBHR1 with BstB I. The resulting plasmids pBHR-MCS1 and pBHR-MCS2 contained the lacZ-cat insert in different orientations; only pBHR-MCS1 was used further. Next, a transcriptional terminator sequence encoded by rrnB was PCR amplified using primers rrnB- Kpn I-fw (5′-TAA GGTACC CGGGGATCCTCTAGAGTCG-3′) and rrnB- Kpn I-rv (5′-CGC GGTACC AAGAGTTTGTAGAAACGCAAA-3′), which both included Kpn I-site overhangs, and plasmid pSCrhaB1  as a template. The 472-bp PCR fragment was digested with Kpn I and cloned into pBHR-MCS1. The correct orientation of the rrnB insert in the resulting plasmid pBHR1-MR
GSK126 research buy was confirmed by PCR using primers rrnB-fw (5′-TCAGAAGTGAAACGCCGTAG-3′) and cat1-rv Regorafenib clinical trial (5′-ACGTGGCCAATATGGACAAC-3′). Next, a synthetic gene encoding a variant of the far-red fluorescent protein TurboFP635 (scientific name Katushka) was obtained from Source BioScience (formerly Geneservice). The variant turboFP635 sequence had been adapted to the codon bias of B. pseudomallei and was preceded by a Spe I site and followed by an EcoR V site. The 810-bp turboFP635 gene was cut from the cloning vector and cloned into EcoR V/Spe I restricted pBHR1-MR, resulting in plasmid Megestrol Acetate pBHR1-RFP. Finally, a 443-bp fragment spanning the upstream region of the groES gene on chromosome I of B. pseudomallei strain K96243 (BPSL2698) was PCR amplified using primers groESprom-fw (5′-CTT GAGCTC GAACGTCGATTCGGACGCAT-3′) and groESprom-rv (5′-GCGG
ACTAGT ATTCACTCCTCTCTTTGATT-3′), which included Sac I and Spe I restriction sites, respectively. The PCR product was cloned into pBHR1-RFP via its Sac I/Spe I sites, resulting in plasmid pBHR1-groS-RFP (KmR, CmR). For use in intracellular replication assays, the kanamycin resistance cassette of plasmid pBHR1 and the derivatives described had to be eliminated by the following method. Firstly, unmethylated pBHR1 plasmid DNA isolated from a dcm -/dam – E. coli strain C2925 (New England Biolabs) was cut with Stu I/PpuM I, which resulted in a 1.2-kb fragment encompassing the kanamycin resistance cassette and a 4.1-kb plasmid backbone fragment. The 4.1-kb fragment was treated with T4 DNA polymerase (Promega) according to the manufacturer’s recommendations and re-ligated overnight at 15°C resulting in plasmid pBHR4 (CmR). Finally, a 1-kb fragment representing the cat gene of plasmid pBHR4 was replaced by a 3.2-kb fragment of plasmid pBHR1-groS-RFP, which encompassed the RFP gene linked to the groES promoter, the rrnB terminator and the cat gene, via BstB I restriction as described for the construction of pBHR-MCS1&2. This resulted in plasmid pBHR4-groS-RFP (CmR).