From e0b0f46918222b3855aaef0b249d57152138a8bb Mon Sep 17 00:00:00 2001
From: Pia Saake <psaake@uni-koeln.de>
Date: Mon, 14 Oct 2024 15:47:14 +0200
Subject: [PATCH] added protocols

---
 assays/BarleyPhenotyping/isa.assay.xlsx       | Bin 6899 -> 11920 bytes
 .../BarleyMaterialAndGrowthConditions.md      |  10 +++++++---
 .../isa.assay.xlsx                            | Bin 22651 -> 22653 bytes
 .../isa.assay.xlsx                            | Bin 19994 -> 19993 bytes
 .../PlasmidConstructionforGBP1Expression.md   |   6 ++++++
 assays/OxidativeBurstAssay/isa.assay.xlsx     | Bin 14460 -> 14459 bytes
 assays/ProteinPull-down/isa.assay.xlsx        | Bin 21251 -> 21250 bytes
 .../isa.study.xlsx                            | Bin 7698 -> 7698 bytes
 studies/HordeumVulgare/isa.study.xlsx         | Bin 15767 -> 15767 bytes
 studies/NicotianaBenthamiana/isa.study.xlsx   | Bin 10602 -> 10602 bytes
 10 files changed, 13 insertions(+), 3 deletions(-)
 create mode 100644 assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstructionforGBP1Expression.md

diff --git a/assays/BarleyPhenotyping/isa.assay.xlsx b/assays/BarleyPhenotyping/isa.assay.xlsx
index e29cfbda77c163adc1bd8f15b27b874cacf46c65..88bc6b72fb8cfbd9aae9e5de1bf7cade622c2d2e 100644
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diff --git a/assays/BarleyPhenotyping/protocols/BarleyMaterialAndGrowthConditions.md b/assays/BarleyPhenotyping/protocols/BarleyMaterialAndGrowthConditions.md
index 3b1a8b9..dbbef43 100644
--- a/assays/BarleyPhenotyping/protocols/BarleyMaterialAndGrowthConditions.md
+++ b/assays/BarleyPhenotyping/protocols/BarleyMaterialAndGrowthConditions.md
@@ -1,7 +1,11 @@
 ## Barley plant material 
 
-All experiments, including the generation of CRISPR/Cas9 knock-out lines, were performed with the spring barley (H. vulgare L.) cv. Golden Promise Fast, an introgression line carrying the Ppd-H1 allele that confers fast flowering. 42 From here on, we use ‘‘control’’ to name this non-mutagenized cultivar that carries normal copies of GBP1 and GBP2. 
+All experiments, including the generation of CRISPR/Cas9 knock-out lines, were performed with the spring barley (*H. vulgare* L.) cv. Golden Promise Fast, an introgression line carrying the *Ppd-H1* allele that confers fast flowering. From here on, we use ‘‘control’’ to name this non-mutagenized cultivar that carries normal copies of *GBP1* and *GBP2*. 
 
-## Barley growth conditions 
+## Barley growth conditions (6 days old) 
 
-Seeds were germinated on wet filter paper at room temperature in the dark under sterile conditions for three days before transfer to sterile jars containing solid 1/10 plant nutrition medium (PNM), pH 5.7 and 0.4% Gelrite (Duchefa, Haarlem, the Netherlands). 82 Seedlings were cultured for four days in a growth chamber under long-day conditions (day/night cycle of 16/8 h, 22 C/18 C, light intensity of 108 mmol$m2$s1 ).
\ No newline at end of file
+Seeds were germinated on wet filter paper at room temperature in the dark under sterile conditions for three days before transfer to sterile jars containing solid 1/10 plant nutrition medium (PNM), pH 5.7 and 0.4% Gelrite (Duchefa, Haarlem, the Netherlands). Seedlings were cultured for six days in a growth chamber under long-day conditions (day/night cycle of 16/8 h, 22 C/18 C, light intensity of 108 mmol$m2$s1). Root and shoot fresh weight as well as length of the barley plants were measured. 
+
+## Barley growth conditions (28 days old) 
+
+Seeds were germinated on wet filter paper at room temperature in the dark under sterile conditions for three days before transfer to pots (15 cm) containing heat-treated soil mixture (Cologne Agricultural Soil:vermiculite; 1:1). Seedlings were grown in the greenhouse for 28 days until harvest. Root and shoot fresh weight as well as length of the barley plants were measured. 
\ No newline at end of file
diff --git a/assays/FungalBarleyColonizationAssays/isa.assay.xlsx b/assays/FungalBarleyColonizationAssays/isa.assay.xlsx
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diff --git a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx
index fdafdab59d805b88259f0617bda54d522fdf87ed..f6fe391962f90902bef7b84566242cd8514499de 100644
GIT binary patch
delta 942
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diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstructionforGBP1Expression.md b/assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstructionforGBP1Expression.md
new file mode 100644
index 0000000..d5d772b
--- /dev/null
+++ b/assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstructionforGBP1Expression.md
@@ -0,0 +1,6 @@
+## 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 50- and 30-UTRs of RNA-2 from the cowpea mosaic virus as translational enhancers, and C-terminal hemagglutinin (HA) and StrepII tags. 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, Du¨ ren, 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 X 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, Du¨ ren, 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/OxidativeBurstAssay/isa.assay.xlsx b/assays/OxidativeBurstAssay/isa.assay.xlsx
index 02f61042b9b6f3e73b7ad121f7332dda52dabea3..1e11732b3bc0c91800dbc7c4f71d52574f86ba94 100644
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diff --git a/assays/ProteinPull-down/isa.assay.xlsx b/assays/ProteinPull-down/isa.assay.xlsx
index 3bdc28d33a19b08eb9150b1eb05be7eed75fd1f6..4c56c0dbefec79c2dfffe6075912aafc81b27fc7 100644
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delta 933
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diff --git a/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx b/studies/CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays/isa.study.xlsx
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diff --git a/studies/HordeumVulgare/isa.study.xlsx b/studies/HordeumVulgare/isa.study.xlsx
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diff --git a/studies/NicotianaBenthamiana/isa.study.xlsx b/studies/NicotianaBenthamiana/isa.study.xlsx
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delta 388
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-- 
GitLab