diff --git a/.gitattributes b/.gitattributes index 51d9aaea27cc000f253db93becf75866ea00ff49..693db821c9296805d5d593081b2fd95f4c502ada 100644 --- a/.gitattributes +++ b/.gitattributes @@ -18,3 +18,13 @@ assays/2-2_ChAP-analysis/dataset/AnalyzedData/analysis_hrrA_iron.7z filter=lfs d assays/3_ReporterAssays/dataset/NCgl0176_NCgl0177_wzy/20240423_BiolectorAK123_Raw.csv filter=lfs diff=lfs merge=lfs -text assays/3_ReporterAssays/dataset/NCgl2776_gyrB_wzy/20240214_BiolectorAK115_Raw.csv filter=lfs diff=lfs merge=lfs -text assays/3_ReporterAssays/dataset/metH-NCgl1451/20240215_BiolectorAK114_Raw.csv filter=lfs diff=lfs merge=lfs -text +assays/4_ElectronMobilityShiftAssays_DtxR/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text +assays/4_ElectronMobilityShiftAssays_DtxR/dataset/EMSA‘s[[:space:]]DtxR.pptx filter=lfs diff=lfs merge=lfs -text +assays/4_ElectronMobilityShiftAssays_DtxR/dataset/Gel2.tiff filter=lfs diff=lfs merge=lfs -text 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0000000000000000000000000000000000000000..214c67960b4294bf5b02eb0c03433fffab931ad2 --- /dev/null +++ b/assays/4_ElectronMobilityShiftAssays_DtxR/dataset/controlgel2.tiff @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:20dd6cc49fc9c0d8ef29115bdb05bc9fdc3ca36bc1b27beed31af51052495124 +size 7303576 diff --git a/assays/4_ElectronMobilityShiftAssays_DtxR/isa.assay.xlsx b/assays/4_ElectronMobilityShiftAssays_DtxR/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..3cd3648035b3b3cc69fedd68377f42b8345c4650 Binary files /dev/null and b/assays/4_ElectronMobilityShiftAssays_DtxR/isa.assay.xlsx differ diff --git a/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/.gitkeep b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md new file mode 100644 index 0000000000000000000000000000000000000000..e43bd3803b4c4db65ba3be3132b853d9e06e5201 --- /dev/null +++ b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md @@ -0,0 +1,2 @@ +The purified DNA fragments of 121 bp size covering the region of identified ChAP-Seq peaks (61 bp peak extended 30 bp to each side; final concentration 31 nM) were incubated +for 30 min at room temperature with 0, 50 or 200-fold purified DtxR protein excess or 0, 300 and 400-fold excess, respectively. The reaction buffer contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), and 150 μM MnCl2. Afterwards,separation followed by a gel electrophoresis using a native polyacrylamide (10%) gel supplemented with 1 mM DTT and 150 μM MnCl2. Electrophoresis was performed at 170 V using TB buffer (89 mM Tris, 89 mM boric acid) supplemented with 1 mM DTT and 150 μM MnCl2 for 1.5 h. Gels were stained for 20 min with ethidium bromide. 121 bp promoter region of acn served as negative control. Protocol was adjusted according to Wennerhold and Bott (2006). \ No newline at end of file diff --git a/studies/4_InVitroBindingOfDtxR/README.md b/studies/4_InVitroBindingOfDtxR/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/4_InVitroBindingOfDtxR/isa.study.xlsx b/studies/4_InVitroBindingOfDtxR/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..e8021b1707aae3a7194bc3bfce4bb45458629b4b Binary files /dev/null and b/studies/4_InVitroBindingOfDtxR/isa.study.xlsx differ diff --git a/studies/4_InVitroBindingOfDtxR/protocols/.gitkeep b/studies/4_InVitroBindingOfDtxR/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md b/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md new file mode 100644 index 0000000000000000000000000000000000000000..521debc7124bd5d1143940753d89f76c292585f6 --- /dev/null +++ b/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md @@ -0,0 +1 @@ +DNA fragments of 121 bp size covering the region of identified ChAP-Seq peaks were generated using respective primer pairs. Purification followed using NucleoSpin Gel and PCR Clean-up Kit (Macherey Nagel, Düren, Germany) eluting in H2O. Concentrations were measured using nanophotometer. \ No newline at end of file diff --git a/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md b/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md new file mode 100644 index 0000000000000000000000000000000000000000..79e1beaeed486a0645fb4769b3a6177d24c40bb6 --- /dev/null +++ b/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md @@ -0,0 +1 @@ +The expression plasmid pET24b-dtxR-C in E. coli BL21(DE3) was used for overproduction of DtxR of Corynebacterium glutamicum containing a His-Tag (HHHHLEHHHHHH) at the carboxyl terminus as previously described (Wennerhold and Bott, 2006). DtxR was purified using Ni-NTA agarose column material (Thermo Fisher Scientific, USA) according to manufacturer’s instructions to the gravity flow protocol. Fractions containing DtxR were pooled and elution buffer was exchanged against TG buffer (30 mM Tris-HCl, 10% (v/v) glycerol, pH 7.5) using PD-10 desalting columns (Cytiva, USA). Concentration was firstly evaluated with Bradford assay, and further measured using Qubit Protein Assay Kit (Invitrogen). \ No newline at end of file diff --git a/studies/4_InVitroBindingOfDtxR/resources/.gitkeep b/studies/4_InVitroBindingOfDtxR/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/4_InVitroBindingOfDtxR/resources/20250224_EMSAPrimer.xlsx b/studies/4_InVitroBindingOfDtxR/resources/20250224_EMSAPrimer.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..86d3070d0f20980d9d2aa58ec9e15c2361ea613d Binary files /dev/null and b/studies/4_InVitroBindingOfDtxR/resources/20250224_EMSAPrimer.xlsx differ diff --git a/studies/4_InVitroBindingOfDtxR/resources/NC_003450.3_peak regions marked.dna b/studies/4_InVitroBindingOfDtxR/resources/NC_003450.3_peak regions marked.dna new file mode 100644 index 0000000000000000000000000000000000000000..4fc0e2c502730d7203f834163971458f694c9b4c --- /dev/null +++ b/studies/4_InVitroBindingOfDtxR/resources/NC_003450.3_peak regions marked.dna @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:37e0e2ff93f508666c289ac5e5c48f6a2db24e052252474053f9414c1449ffc9 +size 15933520