Plasmid DNA was prepared using QIAprep Spin Miniprep Kit (Qiagen

Plasmid DNA was Cediranib in vitro prepared using QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Restriction enzymes were purchased from New England Biolabs (Schwalbach, Germany) or Fermentas (St. Leon-Rot, Germany) and used according to supplier recommendations. Oligonucleotides for PCR were purchased from Thermo Fisher Scientific (Ulm, Germany). DNA sequencing was commissioned to Scientific Research and Development (Bad Homburg, Germany). Polymerase chain reactions (PCR) were performed Inhibitors,research,lifescience,medical as described by [57] using TaKaRa DNA polymerase from Lonza (Köln, Germany). Construction of plasmids. The open reading frame of sgrT (sgrT ORF) was amplified using chromosomal

DNA of LJ110, which is closely related to wild type E. coli K-12, with forward primer sgrT+, containing a PstI restriction site and reverse primer Inhibitors,research,lifescience,medical sgrT-, which had a HindIII restriction site. This PCR product was purified with Wizard DNA purification system (Promega) and cloned into the vector pTM30, resulting in plasmid pTM30sgrT. The expression plasmid pTM30 provides a tac-promoter, an Inhibitors,research,lifescience,medical artificial start codon and and an artificial ribosomal binding site as described before [54]. pTM30sgrT3HA carries additional sequences that encode a 3xHA tag (received from pFA6a3HA, Oligonucleotides HA+/-) fused to the C-terminus of SgrT. For pACYC184sgrT3HA the tacPO and sgrT3HA sequence from pTM30sgrT3HA

was amplified with Inhibitors,research,lifescience,medical oligonucleotides TacPO+ and HA- and cloned into the vector pACYC184. For the bimolecular fluorescence complementation assay the sgrT

ORF was amplified by PCR (Oligonucleotides pETS+/-) and cloned into the vector pET11a-link-NGFP [52] using the restriction enzymes XhoI/BamHI. The genes encoding EIIBGlc (aa 389-477), Linker-EIIBGlc (aa 380-477), Inhibitors,research,lifescience,medical Linker-EIIBGlc-P384R (aa 380-477), EIICGlc (aa 1-381), EIICGlc-Linker (aa 1-396), EIICGlc-Linker-P384R (aa 1-396) and EIICBGlc (aa 1-477) were also amplified by PCR and purified. The oligonucleotides were used as follows: EIIBGlc (pMRB+/pMRG-), Linker-EIIBGlc (pMRLB+/pMRG-), Linker-EIIBGlc-P384R (pMRLB-P384R+/pMRG-), EIICGlc(pMRG+/pMRC-), EIICGlc-Linker (pMRG+/pMRCL-), EIICGlc-Linker-P384R (pMRG+/pMRCL-P384R-) and EIICBGlc (pMRG+/-). PCR products were cloned into the vector pMRBAD-link-CGFP Tryptophan synthase [52] using the restriction enzymes NcoI/AatII or SphI/AatII, respectively. For fluorescence microscopy, an SgrT-GFP fusion protein was created. The sgrT ORF was amplified with oligonucleotides SgrT+/SgrT2- and a gfp gene was amplified using pBLP2 as template and oligonucleotides Gfp2+/-. Both PCR products were purified and cloned together into pTM30, resulting in pTM30sgrT-gfp. All oligonucleotide sequences used are listed in Table3 in the supplemental materials. Site-directed mutagenesis.

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