From 04ddab0770bed0bd95651805eec058674f0fbd43 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Sat, 15 Jun 2024 13:18:14 +0200
Subject: [PATCH] recreate assay
---
.../isa.assay.xlsx | Bin 9199 -> 9199 bytes
.../isa.assay.xlsx | Bin 15148 -> 15148 bytes
.../isa.assay.xlsx | Bin 14945 -> 14945 bytes
.../isa.assay.xlsx | Bin 24268 -> 24268 bytes
assays/MALDI-TOFAnalysis/isa.assay.xlsx | Bin 9838 -> 9838 bytes
.../isa.assay.xlsx | Bin 15096 -> 15096 bytes
.../MultipleSequenceAlignment/isa.assay.xlsx | Bin 6904 -> 6904 bytes
assays/OxidativeBurstAssay/isa.assay.xlsx | Bin 13308 -> 13308 bytes
.../README.md | 0
.../dataset/.gitkeep | 0
.../isa.assay.xlsx | Bin 0 -> 6919 bytes
.../protocols/.gitkeep | 0
.../protocols/PlasmidConstruction.md | 9 +++++++++
assays/ProteinPull-down/isa.assay.xlsx | Bin 17683 -> 17683 bytes
.../isa.assay.xlsx | Bin 6911 -> 6911 bytes
.../isa.assay.xlsx | Bin 6909 -> 6909 bytes
assays/qRT-PCR/isa.assay.xlsx | Bin 9592 -> 9592 bytes
isa.investigation.xlsx | Bin 9052 -> 9052 bytes
.../isa.study.xlsx | Bin 7597 -> 7597 bytes
studies/HordeumVulgare/isa.study.xlsx | Bin 15460 -> 15460 bytes
studies/NicotianaBenthamiana/isa.study.xlsx | Bin 10403 -> 10432 bytes
21 files changed, 9 insertions(+)
create mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/README.md
create mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep
create mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx
create mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep
create mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md
diff --git a/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx b/assays/CRISPRCas9-basedMutagenesis/isa.assay.xlsx
index 722ab5132c7a8040d83db66d10ede10c9d9a1fda..fb257c2c3d9a4105761ff2dd738217077664457e 100644
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diff --git a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx
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diff --git a/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx b/assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.assay.xlsx
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diff --git a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx
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diff --git a/assays/MALDI-TOFAnalysis/isa.assay.xlsx b/assays/MALDI-TOFAnalysis/isa.assay.xlsx
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diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md
new file mode 100644
index 0000000..aa8bbf5
--- /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
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diff --git a/assays/qRT-PCR/isa.assay.xlsx b/assays/qRT-PCR/isa.assay.xlsx
index 45193c6f68644795e896311e87aa1fa3785bbf55..356d284d4804f5d569fe79d10a2022d74bcfe08c 100644
GIT binary patch
delta 687
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delta 687
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index d8584f4287400c72d90da4511cf726a28ac76643..86cdd66c94263b5d68a829153eca1e0a1bf95784 100644
GIT binary patch
delta 611
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delta 611
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diff --git a/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx b/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx
index b28490d189abedb3586cc3c27be75b22f5a6c5f1..2a52c84787bf523c7fb1017f6a97b985a2747629 100644
GIT binary patch
delta 615
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delta 615
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diff --git a/studies/HordeumVulgare/isa.study.xlsx b/studies/HordeumVulgare/isa.study.xlsx
index e7090b29190281ba63a64e7d32e828cb1f8beeb8..8da44b46a0fc841711c0ec5b403902e69885bac5 100644
GIT binary patch
delta 837
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delta 837
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diff --git a/studies/NicotianaBenthamiana/isa.study.xlsx b/studies/NicotianaBenthamiana/isa.study.xlsx
index 67d106d2d8cd12118b075a5f63994d90e4db35c0..7328dc4fa3de23fa4c16fa58e3b76c1202caf3f8 100644
GIT binary patch
delta 3512
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--
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