From fa410189236af3036ce143be322d082d996f78e9 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Sat, 30 Dec 2023 18:58:52 +0100
Subject: [PATCH] add ProteinProductionAndPurification assay
---
.../isa.assay.xlsx | Bin 6915 -> 6915 bytes
.../README.md | 0
.../dataset/.gitkeep | 0
.../isa.assay.xlsx | Bin 0 -> 6916 bytes
.../protocols/.gitkeep | 0
...ologousProteinProductionAndPurification.md | 2 ++
assays/MALDI-TOFAnalysis/isa.assay.xlsx | Bin 6901 -> 6901 bytes
.../isa.assay.xlsx | Bin 7021 -> 7021 bytes
assays/OxidativeBurstAssay/isa.assay.xlsx | Bin 6900 -> 6900 bytes
.../isa.assay.xlsx | Bin 6911 -> 6911 bytes
assays/qRT-PCR/isa.assay.xlsx | Bin 6894 -> 6894 bytes
isa.investigation.xlsx | Bin 11582 -> 11616 bytes
studies/Barley/isa.study.xlsx | Bin 7781 -> 7781 bytes
.../isa.study.xlsx | Bin 7536 -> 7536 bytes
studies/NicotianaBenthamiana/isa.study.xlsx | Bin 7506 -> 7550 bytes
.../isa.study.xlsx | Bin 7518 -> 7518 bytes
16 files changed, 2 insertions(+)
create mode 100644 assays/HeterologousProteinProductionAndPurification/README.md
create mode 100644 assays/HeterologousProteinProductionAndPurification/dataset/.gitkeep
create mode 100644 assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx
create mode 100644 assays/HeterologousProteinProductionAndPurification/protocols/.gitkeep
create mode 100644 assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md
diff --git a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx
index 3f06afef11bc5fd0acf1227f5a332ce28cd74944..fdc14a22e6e13bdd67e5725adec461718cf41fe2 100644
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new file mode 100644
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new file mode 100644
index 0000000..e69de29
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diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/.gitkeep b/assays/HeterologousProteinProductionAndPurification/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md
new file mode 100644
index 0000000..7efd982
--- /dev/null
+++ b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md
@@ -0,0 +1,2 @@
+## Heterologous protein production and purification from N. benthamiana
+*A. tumefaciens* GV3101::pMP90RK strains carrying the binary vectors for protein production (antibiotic selection: 30 μg·mL-1 Rifampicin, 25 μg·mL-1 Kanamycin, 50 μg·mL-1 Carbenicillin) and *A. tumefaciens* GV3101 strains carrying the binary vector for viral p19 silencing inhibitor expression (antibiotic selection: 30 μg·mL-1 Rifampicin, 30 μg·mL-1 Gentamicin, 100 μg·mL-1 Carbenicillin) were grown in selection LB liquid medium at 28 °C, 180 rpm for three days. The cultures were centrifuged (3,500 g for 15 min), resuspended in infiltration buffer (10 mM MES pH 5.5, 10 mM MgCl2, 200 μM acetosyringone) to an OD600 of 1 and incubated for 1 h in the dark at 28 °C, 180 rpm. Each of the two *A. tumefaciens* strains carrying the GBP1 production constructs was mixed with the *A. tumefaciens* strain carrying the p19-expressing construct in a 1:1 ratio. The bacterial suspensions were infiltrated into the four youngest, fully developed leaves of four-week-old *N. benthamiana* plants with a needleless syringe. Five days after infiltration, the leaves were detached from the plant and ground in liquid nitrogen. Protein purification was carried out according to Werner and coworkers84 with minor modifications: The ground plant material (up to the 5 mL mark of 15-mL tube) was thoroughly resuspended in 5 mL of ice-cold extraction buffer (100 mM Tris pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 10 mM DTT, 100 μg·mL-1 Avidin) and centrifuged at 10,000 g, 4 °C for 10 min. The supernatant was filtered through a PD-10 desalting column (Sigma-Aldrich, Taufkirchen, Germany), transferred to a new tube, and supplemented with 75 μL·mL-1 Strep-Tactin Macroprep (50% slurry) (IBA Lifesciences GmbH, Göttingen, Germany). Samples were incubated in a rotary wheel at 4 °C for 1 h, followed by centrifugation for 30 s at 700 g. The supernatant was discarded, and the beads were washed three times with 2 mL of washing buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.005% Triton X-100, 2 mM DTT). Proteins were eluted from the beads by adding 100 μL of elution buffer (wash buffer containing 10 mM biotin) and incubating at 800 rpm for 5 min at 25 °C. The samples were centrifuged at 700 g for 20 s and the elution was repeated two more times. The elution fractions were pooled and dialyzed overnight against cold Milli-Q water (dialysis tubing with 6-8 kDa cut-off). Proteins were stored on ice at 4 °C for further use. The success of protein purification was analyzed by SDS PAGE and Western Blotting.
\ No newline at end of file
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diff --git a/studies/Barley/isa.study.xlsx b/studies/Barley/isa.study.xlsx
index e66c2ddd120d452162c8cc2f05df705a9f1549aa..3b88dfa48a1092ebc4f975a42dd6918f5d37a3d3 100644
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diff --git a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx
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diff --git a/studies/NicotianaBenthamiana/isa.study.xlsx b/studies/NicotianaBenthamiana/isa.study.xlsx
index 989f05098c9f5b33ef1d701539b5c5fe39911ba3..191640abad08d48c42764ba3637a46350566a9d8 100644
GIT binary patch
delta 2079
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diff --git a/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx
index 438c42b241eb16534b10c2217e9d0950499862e1..7b3a4866c50feea27cf08bae22b6925e28709f08 100644
GIT binary patch
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delta 526
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--
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