From c8c6a340815d3f00febe7fdc02a1d00b9b1e55a3 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Sat, 15 Jun 2024 13:16:55 +0200
Subject: [PATCH] delete assay

---
 .../README.md                                   |   0
 .../dataset/.gitkeep                            |   0
 .../isa.assay.xlsx                              | Bin 24267 -> 0 bytes
 .../protocols/.gitkeep                          |   0
 .../protocols/PlasmidConstruction.md            |   9 ---------
 5 files changed, 9 deletions(-)
 delete mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/README.md
 delete mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep
 delete mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx
 delete mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep
 delete mode 100644 assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md

diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/README.md b/assays/PlasmidPreparationForHeterologousProteinProduction/README.md
deleted file mode 100644
index e69de29..0000000
diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep b/assays/PlasmidPreparationForHeterologousProteinProduction/dataset/.gitkeep
deleted file mode 100644
index e69de29..0000000
diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx b/assays/PlasmidPreparationForHeterologousProteinProduction/isa.assay.xlsx
deleted file mode 100644
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diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/.gitkeep
deleted file mode 100644
index e69de29..0000000
diff --git a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md b/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md
deleted file mode 100644
index aa8bbf5..0000000
--- a/assays/PlasmidPreparationForHeterologousProteinProduction/protocols/PlasmidConstruction.md
+++ /dev/null
@@ -1,9 +0,0 @@
-## 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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