diff --git a/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx b/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx index 722ab5132c7a8040d83db66d10ede10c9d9a1fda..fb257c2c3d9a4105761ff2dd738217077664457e 100644 Binary files a/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx and b/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx differ diff --git a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx index cbcda07a36aaa28e5c68b772942e8e83b397c1e3..9f965dfb6f684f6075a7421ab89ed2be4035680d 100644 Binary files a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx and b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx differ diff --git a/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx b/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx index 3204ee06c8f3a93e3f9546479c8278c4ac82b2d5..6e3d7bcce2f35175ec77551525201c1db9ba1850 100644 Binary files a/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx and b/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx differ diff --git a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx index 27eb9d2350a7e2cf1daaaad55c1b0abf73cd072c..27fce6b30fa9cbcdf735a98a29e4543958afe9a2 100644 Binary files a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx and b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx differ diff --git a/assays/MALDI-TOFAnalysis/isa.assay.xlsx b/assays/MALDI-TOFAnalysis/isa.assay.xlsx index 18440b6743f238ce3bdb68e3faab566373f95f5e..f82f1410623f344deac5f958a149634f49c6bed8 100644 Binary files a/assays/MALDI-TOFAnalysis/isa.assay.xlsx and b/assays/MALDI-TOFAnalysis/isa.assay.xlsx differ diff --git a/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx b/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx index 11ca86226a265a28a953fae2a4db9e6dab47cf0d..f940d5b81480614e62375708e3a208afc8caf849 100644 Binary files a/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx and b/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx differ diff --git a/assays/MultipleSequenceAlignment/isa.assay.xlsx b/assays/MultipleSequenceAlignment/isa.assay.xlsx index 6fe6daa91d06741eb249c20e22506ce00cf6c52a..133202e7dd6bac596cd63ac9fce60ba0bb9a16e4 100644 Binary files a/assays/MultipleSequenceAlignment/isa.assay.xlsx and b/assays/MultipleSequenceAlignment/isa.assay.xlsx differ diff --git a/assays/OxidativeBurstAssay/isa.assay.xlsx b/assays/OxidativeBurstAssay/isa.assay.xlsx index df714edce5c8f85eb81aacd634172515ea3b39ee..fdb2347c3b27d0e82644e9b3ec8492fca7de1ffa 100644 Binary files a/assays/OxidativeBurstAssay/isa.assay.xlsx and b/assays/OxidativeBurstAssay/isa.assay.xlsx differ diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/README.md b/assays/PlasmidPreparationForHeterologousProteinProduction/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep b/assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx b/assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..9a3ab7548808856ad65af80ae6f63fb655646daa Binary files /dev/null and b/assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx differ diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md new file mode 100644 index 0000000000000000000000000000000000000000..aa8bbf596c2b4645982747b902374264c7ec01f5 --- /dev/null +++ b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md @@ -0,0 +1,9 @@ +## Plasmid construction for the heterologous expression of barley GBP1 in *N. benthamiana* + +For *in planta* protein production in *N. benthamiana*, we used the binary vector pXCScpmv-HAStrep characterized by a 35S promoter cassette, modified 5′- and 3′-UTRs of RNA-2 from the cowpea mosaic virus as translational enhancers, and C-terminal hemagglutinin (HA) and StrepII tags (Witte et al., 2004; Myrach et al., 2017). The codon-optimized GBP1 coding sequence was amplified with the primer pair ClaI_GBP1_F (5’-gacggtatcgataaaATGCCGCCACATGGTAGACG-3’) and GBP1_noSTOP_XmaI_R (5’-ataactcccgggATGGCCATATTGACGATACCAACAGC-3’) and directionally cloned into the ClaI and XmaI sites of the binary vector to produce pXCScpmv-GBP1-HAStrep. + +To generate a catalytically inactive version of GBP1, the first glutamate residue of the catalytic center (E500) was exchanged to an alanine residue via site-directed mutagenesis PCR with the primer pair GBP1_E500A_F (5’-CAGGCATCAACATCAGAAGCAGTG-3’) and GBP1_E500A_R (5’-GTTCCTACCATCTCCAAACTCAGTC-3’). + +The linearized, mutated plasmid was purified after gel electrophoresis using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The isolated DNA fragment was treated with a self-made KLD mixture (1,000 units·mL-1 T4 polynucleotide kinase, 40,000 T4 DNA ligase units·mL-1 ligase 2,000 units·mL-1 DpnI, 1 × T4 DNA ligase buffer; all enzymes were purchased from New England BioLabs, Ipswich, USA) for 1 h at room temperature before transformation into *Escherichia coli* MachI cells. + +Plasmids were isolated using the NucleoSpin Plasmid Kit (Machery-Nagel, Düren, Germany) and sequenced to confirm the introduced mutation. Both plasmids (pXCScpmv-GBP1-HAStrep and pXCScpmv-GBP1_E500A-HAStrep) were introduced into *Agrobacterium tumefaciens* GV3101::pMP90RK strains for transient transformation of *N. benthamiana* leaf tissue. \ No newline at end of file diff --git a/assays/ProteinPull-down/isa.assay.xlsx b/assays/ProteinPull-down/isa.assay.xlsx index ac19c6d765e6edf6db93906a5df09364cd47476f..0b2299ad5418f94da9de30eb68c4c71e5f40b674 100644 Binary files a/assays/ProteinPull-down/isa.assay.xlsx and b/assays/ProteinPull-down/isa.assay.xlsx differ diff --git a/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx b/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx index c39c95fb9be6d4a2283aee6501dc009239f15c0f..717a6eb839f9d6bcd72b9ddb538b9ffcddc334a9 100644 Binary files a/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx and b/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx differ diff --git a/assays/StainingForConfocalMicroscopy/isa.assay.xlsx b/assays/StainingForConfocalMicroscopy/isa.assay.xlsx index 10bca5e7af21cf9f1db3b20b0ca62f9ccb33c29a..a016ebb6fd429e9a4566fc630a8e78fbf27e321a 100644 Binary files a/assays/StainingForConfocalMicroscopy/isa.assay.xlsx and b/assays/StainingForConfocalMicroscopy/isa.assay.xlsx differ diff --git a/assays/qRT-PCR/isa.assay.xlsx b/assays/qRT-PCR/isa.assay.xlsx index 45193c6f68644795e896311e87aa1fa3785bbf55..356d284d4804f5d569fe79d10a2022d74bcfe08c 100644 Binary files a/assays/qRT-PCR/isa.assay.xlsx and b/assays/qRT-PCR/isa.assay.xlsx differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index d8584f4287400c72d90da4511cf726a28ac76643..86cdd66c94263b5d68a829153eca1e0a1bf95784 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx b/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx index b28490d189abedb3586cc3c27be75b22f5a6c5f1..2a52c84787bf523c7fb1017f6a97b985a2747629 100644 Binary files a/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx and b/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx differ diff --git a/studies/HordeumVulgare/isa.study.xlsx b/studies/HordeumVulgare/isa.study.xlsx index e7090b29190281ba63a64e7d32e828cb1f8beeb8..8da44b46a0fc841711c0ec5b403902e69885bac5 100644 Binary files a/studies/HordeumVulgare/isa.study.xlsx and b/studies/HordeumVulgare/isa.study.xlsx differ diff --git a/studies/NicotianaBenthamiana/isa.study.xlsx b/studies/NicotianaBenthamiana/isa.study.xlsx index 67d106d2d8cd12118b075a5f63994d90e4db35c0..7328dc4fa3de23fa4c16fa58e3b76c1202caf3f8 100644 Binary files a/studies/NicotianaBenthamiana/isa.study.xlsx and b/studies/NicotianaBenthamiana/isa.study.xlsx differ