From fef859cfb9b082463edcc4a9f39b406128b6d177 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Sun, 6 Oct 2024 21:15:36 +0200
Subject: [PATCH 01/35] License file

---
 LICENSE | 0
 1 file changed, 0 insertions(+), 0 deletions(-)
 create mode 100644 LICENSE

diff --git a/LICENSE b/LICENSE
new file mode 100644
index 0000000..e69de29
-- 
GitLab


From 5021b27336a0b114aba361f715c32016134734f5 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Sun, 6 Oct 2024 21:22:56 +0200
Subject: [PATCH 02/35] Added Supplemental files

---
 .gitattributes                        | 1 +
 LICENSE                               | 0
 _publication/pnas.2221508120.sapp.pdf | 3 +++
 3 files changed, 4 insertions(+)
 delete mode 100644 LICENSE
 create mode 100644 _publication/pnas.2221508120.sapp.pdf

diff --git a/.gitattributes b/.gitattributes
index 14291ea..a888e4c 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -1,2 +1,3 @@
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+_publication/pnas.2221508120.sapp.pdf filter=lfs diff=lfs merge=lfs -text
diff --git a/LICENSE b/LICENSE
deleted file mode 100644
index e69de29..0000000
diff --git a/_publication/pnas.2221508120.sapp.pdf b/_publication/pnas.2221508120.sapp.pdf
new file mode 100644
index 0000000..087d73c
--- /dev/null
+++ b/_publication/pnas.2221508120.sapp.pdf
@@ -0,0 +1,3 @@
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+size 2564675
-- 
GitLab


From 1b7f4730c9a66cdbeae76ccd367285445108d7d5 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Sun, 6 Oct 2024 22:05:49 +0200
Subject: [PATCH 03/35] added supplementary tables

---
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 _publication/dataverse_files/MANIFEST.TXT  |   5 +++++
 _publication/dataverse_files/Table S1.xlsx | Bin 0 -> 179712 bytes
 _publication/dataverse_files/Table S2.xlsx |   3 +++
 _publication/dataverse_files/Table S3.xlsx | Bin 0 -> 27799 bytes
 _publication/dataverse_files/Table S4.xlsx | Bin 0 -> 16339 bytes
 _publication/dataverse_files/Table S5.xlsx | Bin 0 -> 13477 bytes
 7 files changed, 9 insertions(+)
 create mode 100644 _publication/dataverse_files/MANIFEST.TXT
 create mode 100644 _publication/dataverse_files/Table S1.xlsx
 create mode 100644 _publication/dataverse_files/Table S2.xlsx
 create mode 100644 _publication/dataverse_files/Table S3.xlsx
 create mode 100644 _publication/dataverse_files/Table S4.xlsx
 create mode 100644 _publication/dataverse_files/Table S5.xlsx

diff --git a/.gitattributes b/.gitattributes
index a888e4c..7cc7aff 100644
--- a/.gitattributes
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@@ -1,3 +1,4 @@
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 _publication/pnas.2221508120.sapp.pdf filter=lfs diff=lfs merge=lfs -text
+_publication/dataverse_files/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
diff --git a/_publication/dataverse_files/MANIFEST.TXT b/_publication/dataverse_files/MANIFEST.TXT
new file mode 100644
index 0000000..6060df6
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-- 
GitLab


From 193617984fc9b810f8deed6f8b94e48edd6fd62b Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 10 Oct 2024 17:28:28 +0200
Subject: [PATCH 04/35] Adding Study Protocol and Resources (Tables)

---
 studies/Bacterial culture conditions/README.md |   0
 .../isa.study.xlsx                             | Bin 0 -> 7511 bytes
 .../protocols/.gitkeep                         |   0
 .../protocols/BacterialCultureConditions.md    |   3 +++
 .../protocols/BacterialStrains.md              |   3 +++
 .../resources/.gitkeep                         |   0
 .../resources/Table S1.xlsx                    | Bin 0 -> 179712 bytes
 .../resources/Table S3.xlsx                    | Bin 0 -> 27799 bytes
 8 files changed, 6 insertions(+)
 create mode 100644 studies/Bacterial culture conditions/README.md
 create mode 100644 studies/Bacterial culture conditions/isa.study.xlsx
 create mode 100644 studies/Bacterial culture conditions/protocols/.gitkeep
 create mode 100644 studies/Bacterial culture conditions/protocols/BacterialCultureConditions.md
 create mode 100644 studies/Bacterial culture conditions/protocols/BacterialStrains.md
 create mode 100644 studies/Bacterial culture conditions/resources/.gitkeep
 create mode 100644 studies/Bacterial culture conditions/resources/Table S1.xlsx
 create mode 100644 studies/Bacterial culture conditions/resources/Table S3.xlsx

diff --git a/studies/Bacterial culture conditions/README.md b/studies/Bacterial culture conditions/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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diff --git a/studies/Bacterial culture conditions/protocols/.gitkeep b/studies/Bacterial culture conditions/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/Bacterial culture conditions/protocols/BacterialCultureConditions.md b/studies/Bacterial culture conditions/protocols/BacterialCultureConditions.md
new file mode 100644
index 0000000..11aa508
--- /dev/null
+++ b/studies/Bacterial culture conditions/protocols/BacterialCultureConditions.md	
@@ -0,0 +1,3 @@
+## Bacterial culture conditions
+
+Bacteria were streaked from glycerol stocks (25% glycerol) on TSA plates (15 g/l Tryptic Soy Broth, Sigma Aldrich; with 10 g/l Bacto Agar, Duchefa Biochemie) and grown at 25 °C. Single colonies were inoculated into liquid 50% TSB (15 g/l Tryptic Soy broth, Sigma Aldrich) and grown until dense at 25 °C with 180 rpm agitation. Dense cultures were then stored at 4 °C and diluted 1 to 10 in TSB the day before the experiment and cultured at 25 °C with 180 rpm agitation overnight to ensure sufficient cell densities for slow- and rapidly growing bacteria. Glycerol stocks were stored at -80 °C and kept on dry ice when transported.
\ No newline at end of file
diff --git a/studies/Bacterial culture conditions/protocols/BacterialStrains.md b/studies/Bacterial culture conditions/protocols/BacterialStrains.md
new file mode 100644
index 0000000..6a7fb1f
--- /dev/null
+++ b/studies/Bacterial culture conditions/protocols/BacterialStrains.md	
@@ -0,0 +1,3 @@
+## Bacterial Strains
+
+The bacterial strains used in this study have been initially isolated from unplanted soil, *A. thaliana* roots or shoots and are summarized in **Table S1**. *Ralstonia solanacearum* GMI1000 and GMI1600 have also been reported previously. All mutants that were generated in the R401 or R569 backgrounds in this study have been deposited at the Max Planck Institute for Plant Breeding Research and are listed in **Table S3**.
\ No newline at end of file
diff --git a/studies/Bacterial culture conditions/resources/.gitkeep b/studies/Bacterial culture conditions/resources/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/Bacterial culture conditions/resources/Table S1.xlsx b/studies/Bacterial culture conditions/resources/Table S1.xlsx
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-- 
GitLab


From acfd68b07782c4a7f5f71652311138d00447b51b Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 10 Oct 2024 17:50:53 +0200
Subject: [PATCH 05/35] Isa Table for Bacterial Culture

---
 .../isa.study.xlsx                            | Bin 7511 -> 12306 bytes
 1 file changed, 0 insertions(+), 0 deletions(-)

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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-- 
GitLab


From 14b9aaf3f3358e60a0f19b6286c08d609a084cc9 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 10 Oct 2024 18:15:26 +0200
Subject: [PATCH 06/35] ISA table for PlantGrowthConditions - Sterilization

---
 .../isa.study.xlsx                            | Bin 12306 -> 12306 bytes
 studies/Plant Growth Conditions/README.md     |   0
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 0 -> 11175 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/PlantGrowthConditions.md        |   4 ++++
 .../resources/.gitkeep                        |   0
 6 files changed, 4 insertions(+)
 create mode 100644 studies/Plant Growth Conditions/README.md
 create mode 100644 studies/Plant Growth Conditions/isa.study.xlsx
 create mode 100644 studies/Plant Growth Conditions/protocols/.gitkeep
 create mode 100644 studies/Plant Growth Conditions/protocols/PlantGrowthConditions.md
 create mode 100644 studies/Plant Growth Conditions/resources/.gitkeep

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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diff --git a/studies/Plant Growth Conditions/protocols/.gitkeep b/studies/Plant Growth Conditions/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/Plant Growth Conditions/protocols/PlantGrowthConditions.md b/studies/Plant Growth Conditions/protocols/PlantGrowthConditions.md
new file mode 100644
index 0000000..bea2344
--- /dev/null
+++ b/studies/Plant Growth Conditions/protocols/PlantGrowthConditions.md	
@@ -0,0 +1,4 @@
+## Plant Growth Conditions
+
+
+A. thaliana Col-0 wild-type (N60000) was obtained from the Nottingham Arabidopsis Stock Centre (NASC). *Arabidopsis thaliana* Col-0 seeds were sterilized using 70% ethanol and bleach. Seeds were submerged in 70% ethanol and left shaking at 40 rpm for 14 minutes. Ethanol was removed before the seeds were submerged in 8.3% sodium hypochlorite (Roth) containing 1 μl of Tween 20 (Sigma-Aldrich) and left shaking at 40 rpm for 4 minutes. Under sterile conditions, the seeds were washed 7x times and finally taken up with sterile 10 mM MgC12. Seeds were left for stratification at 4 °C for 3 days. Seed sterility was confirmed by plating approx. 100 seeds on a 50% TSA plate.
\ No newline at end of file
diff --git a/studies/Plant Growth Conditions/resources/.gitkeep b/studies/Plant Growth Conditions/resources/.gitkeep
new file mode 100644
index 0000000..e69de29
-- 
GitLab


From 56981b4bc9769d0145c67a59bf5699698cd9dd04 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Mon, 28 Oct 2024 12:48:14 +0100
Subject: [PATCH 07/35] Plant growth conditions

---
 .../isa.study.xlsx                            | Bin 12306 -> 12306 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 11175 -> 15939 bytes
 .../protocols/Agar-media.md                   |   5 +++++
 .../protocols/Flowpot.md                      |   3 +++
 4 files changed, 8 insertions(+)
 create mode 100644 studies/Plant Growth Conditions/protocols/Agar-media.md
 create mode 100644 studies/Plant Growth Conditions/protocols/Flowpot.md

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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
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diff --git a/studies/Plant Growth Conditions/protocols/Agar-media.md b/studies/Plant Growth Conditions/protocols/Agar-media.md
new file mode 100644
index 0000000..83cfaa6
--- /dev/null
+++ b/studies/Plant Growth Conditions/protocols/Agar-media.md	
@@ -0,0 +1,5 @@
+## Agar-media
+
+Surface-sterilized and stratified *A. thaliana* Col-0 seeds were sown on plates containing
+1% agar (Bacto Agar, Difco) in 1⁄2 MS medium supplemented with 0.5% sucrose and placed
+vertically in a climate chamber (Panasonic, MLR-352) and grown for six days (10 h light, 21 °C; 14 h dark, 19 °C). Using a forceps, uniform seedlings were then transferred to freshly prepared 1⁄2 MS plates without sucrose.
\ No newline at end of file
diff --git a/studies/Plant Growth Conditions/protocols/Flowpot.md b/studies/Plant Growth Conditions/protocols/Flowpot.md
new file mode 100644
index 0000000..4fb1763
--- /dev/null
+++ b/studies/Plant Growth Conditions/protocols/Flowpot.md	
@@ -0,0 +1,3 @@
+## Flowpot
+
+Flowpots were assembled and inoculated as described below. Each Flowpot was first flushed with 50 ml sterile MiliQ water and then 50 ml half strength Murashige and Skoog medium with vitamins (1⁄2 MS; 2.2 g/l, Duchefa Biochemie, 0.5 g/l MES, pH 5.7) containing the bacterial inoculum. Per Flowpot, five surface-sterilized and stratified *A. thaliana* Col-0 seeds were pipetted. Microboxes were then incubated in a light cabinet under short day conditions (10 h light at 21 °C, 14 h dark at 19 °C) for 14 days and randomized every 2–3 days.
\ No newline at end of file
-- 
GitLab


From 803b4703e4002679530041cfc51d33c9ef905d2a Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Wed, 13 Nov 2024 13:38:29 +0100
Subject: [PATCH 08/35] new assay Screen for antagonistic interbacterial
 interactions

---
 .gitattributes                                |   2 ++
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../dataset/Table S2.xlsx                     |   3 +++
 .../dataset/pnas.2221508120fig01.jpg          |   3 +++
 .../isa.assay.xlsx                            | Bin 0 -> 7499 bytes
 .../protocols/.gitkeep                        |   0
 .../AntagonisticInterbacterialInteractions.md |   3 +++
 isa.investigation.xlsx                        | Bin 11349 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12306 -> 12305 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15939 bytes
 11 files changed, 11 insertions(+)
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/README.md
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/dataset/.gitkeep
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/dataset/Table S2.xlsx
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/dataset/pnas.2221508120fig01.jpg
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/protocols/.gitkeep
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md

diff --git a/.gitattributes b/.gitattributes
index 7cc7aff..23b3b4c 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -2,3 +2,5 @@
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 _publication/dataverse_files/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
+assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:space:]]interactions/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
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new file mode 100644
index 0000000..e69de29
diff --git a/assays/Screen for antagonistic interbacterial interactions/dataset/.gitkeep b/assays/Screen for antagonistic interbacterial interactions/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
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new file mode 100644
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diff --git a/assays/Screen for antagonistic interbacterial interactions/protocols/.gitkeep b/assays/Screen for antagonistic interbacterial interactions/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md b/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md
new file mode 100644
index 0000000..22b90fd
--- /dev/null
+++ b/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md	
@@ -0,0 +1,3 @@
+## Screen for antagonistic interbacterial interactions
+
+For the initial mBA experiment (**Fig. 1**), bacterial strains were cultured for seven days in 25% TSB (7.5 g/l Tryptic Soy Broth, Sigma-Aldrich). Briefly, 100 μl of a bacterial solution were resuspended in 50 ml cooled (~38 oC), but still molten, 25% TSA (15 g/l Bacto-Agar, Duchefa Biochemie) and poured into a square petri dish (120x120 mm). After medium solidification, 24 bacterial isolates were spotted on top of the medium using a multi-stamp replicator. The replicator was sterilized by dipping in 70% EtOH (v/v) followed by flaming and cooling. The screen comprising 39,204 binary interactions was conducted once and validated by randomly re-screening 7,470 interaction pairs as described above. All bacterial strains that showed antagonistic activity were rescreened two more times. For the *Ralstonia* inhibition screen, *R. solanacearum* GMI1000 was pre-cultured for two days in CPG medium (1% peptone, 0.5% glucose and 0.1% casamino acids; pH7.0). Before spotting the bacterial cultures, each CPG agar plate (1% Peptone, 0.5% D-glucose, 0.1% casamino acids and 1.5% agar; pH 7.0) was overlaid with 5 ml of *R. solanacearum* suspension (50 μl of pre-cultured *R. solanacearum* in pure sterile water). Excess *R. solanacearum* suspension was removed and the plates were briefly dried, then 5 μl of the bacterial culture were spotted onto the *R. solanacearum*-overlaid plates. All isolates were tested three times. For all subsequent halo assays, strains were cultivated in 50% TSB until turbidity, stored at 4 °C and diluted 1:10 in 50% TSB one day before the experiment. Bacterial cultures were pelleted at 4,000 rpm for 15 min. The resulting bacterial pellets were subsequently washed 3 times and resuspended in 1 ml 10 mM MgCl2. OD600 were measured and set depending on the strain. One hundred microliters bacterial culture were inoculated per 50 ml 25% TSA. After drying, up to nine different 3-μl droplets of bacterial suspensions with 0.4 OD600 were applied with equal distances. For all experiments, plates were incubated at 25 °C for up to 96 hours and photographs were taken thereafter for quantitative image analysis. The size of the halo of inhibition was measured using ImageJ with up to five separate measurements, which were subsequently averaged to reduce variation. Raw data of Fig. 1 are indicated in **Table 2**.
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
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-- 
GitLab


From db42026d1c6c67333ee2ac765cd09c1f36986a86 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 31 Jan 2025 11:34:18 +0100
Subject: [PATCH 09/35] added assays and studies

---
 .../isa.assay.xlsx                            | Bin 7499 -> 6920 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7875 bytes
 .../isa.study.xlsx                            | Bin 12305 -> 12307 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15938 bytes
 4 files changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 49fcf1250fda1ce318ddcbd806d4a6e6f4e4c7a5..df0d394cad42d1bd0c873c4428c4af0d2fd65629 100644
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-- 
GitLab


From 81433f8b46b06212eaf4073860029ae36218c959 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Wed, 5 Feb 2025 14:53:51 +0100
Subject: [PATCH 10/35] Added assay for mBA experiment, fig1

---
 .../dataset/mBA experiment.png                |   3 +++
 .../dataset/pnas.2221508120fig01.jpg          |   3 ---
 .../isa.assay.xlsx                            | Bin 6920 -> 9460 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12307 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15938 -> 15939 bytes
 6 files changed, 3 insertions(+), 3 deletions(-)
 create mode 100644 assays/Screen for antagonistic interbacterial interactions/dataset/mBA experiment.png
 delete mode 100644 assays/Screen for antagonistic interbacterial interactions/dataset/pnas.2221508120fig01.jpg

diff --git a/assays/Screen for antagonistic interbacterial interactions/dataset/mBA experiment.png b/assays/Screen for antagonistic interbacterial interactions/dataset/mBA experiment.png
new file mode 100644
index 0000000..859290d
--- /dev/null
+++ b/assays/Screen for antagonistic interbacterial interactions/dataset/mBA experiment.png	
@@ -0,0 +1,3 @@
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diff --git a/assays/Screen for antagonistic interbacterial interactions/dataset/pnas.2221508120fig01.jpg b/assays/Screen for antagonistic interbacterial interactions/dataset/pnas.2221508120fig01.jpg
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index 492e876..0000000
--- a/assays/Screen for antagonistic interbacterial interactions/dataset/pnas.2221508120fig01.jpg	
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F{{TJ|6IcKM

delta 2881
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Bn|%NP

diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index 207a54d11650e32a11060a623d4459bad62d26d1..7cb4cae0c5cf802a64f2339b1fdc28538899b8b3 100644
GIT binary patch
delta 691
zcmX?Xd&HJEz?+$ci-CcGgJDgc>qg#oMrI&Ac{-yah~B)Lksl&(mPrmQ@s3FaEFsD)
z2azyi(F6-5v3zC*Ga@)-S-?swcpysW^Xy^+%ftxJV*<<k6@|#Ch;3%%1X>f-{(Y`>
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gfN3T<Yow&$CI_*tUQU`Zdh%>JCAJq*Ko>Fq0P5NsrvLx|

delta 691
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e%yQO9Nx@wXVq1fpG-K4{IdV#D&!vD)WB>r}zZkOs

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 74e1d2f76270d3e70350c3b883f099b87758b9ab..4494a085727022919adf6ba46033d9b119d9bc05 100644
GIT binary patch
delta 833
zcmbQ7FeQOEz?+$ci-CcGgJDgc>qg#XjLblK@-{|A5WV>pBR@po4U-&Lf|pqZEMdZ|
z1CdB((F6<hvy`)d8JwIqnZQb)a_fU>ah~@O3Eoc-iGTbbzycoy`oUTzH;bx*1y+l$
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zc7FZa6$?YouCkb5BW&|jOd@Oc{*`+#O+ED@a^1tgB*x0w@0{g#9=>i~@a7Wphm8!!
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zN*}B}ck|Ah@%`oF%T=DARJ_s>r?gzmk@dfP)5JH=e)c^5SMG;5)jzr4r`}w*ue*Nc
ze<qX!6ZOB$=6@?A1H&IC1_pj$GOEbY-(00J7m_xlwCupK7_DUpru((b!1M_%Juoe;
zFFjdATN2DR&~^pW721|ydW*I*nEnN&9dzu$(VST|<H*Lz^17mv=js@PRb1AAsFc+8
z1M{<V?ZNaKT~jdq1j<*_gNTD{ZPtU>I$sas!UuX5V71cvCSW>9-x?`VP1A?C`@Ft1
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delta 833
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-- 
GitLab


From 55c46b0b5986883a29af49491da38edb5ea9a4d2 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Wed, 5 Feb 2025 15:30:45 +0100
Subject: [PATCH 11/35] metabolomics protocols

---
 .gitattributes                                |   1 -
 assays/MetabolomicAnalysis/README.md          |   0
 assays/MetabolomicAnalysis/dataset/.gitkeep   |   0
 .../dataset/Metabolomics.md                   |   0
 assays/MetabolomicAnalysis/isa.assay.xlsx     | Bin 0 -> 8680 bytes
 assays/MetabolomicAnalysis/protocols/.gitkeep |   0
 .../protocols/MetabolomicAnalysis.md          |   4 ++++
 .../isa.assay.xlsx                            | Bin 9460 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15939 bytes
 11 files changed, 4 insertions(+), 1 deletion(-)
 create mode 100644 assays/MetabolomicAnalysis/README.md
 create mode 100644 assays/MetabolomicAnalysis/dataset/.gitkeep
 create mode 100644 assays/MetabolomicAnalysis/dataset/Metabolomics.md
 create mode 100644 assays/MetabolomicAnalysis/isa.assay.xlsx
 create mode 100644 assays/MetabolomicAnalysis/protocols/.gitkeep
 create mode 100644 assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md

diff --git a/.gitattributes b/.gitattributes
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diff --git a/assays/MetabolomicAnalysis/README.md b/assays/MetabolomicAnalysis/README.md
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new file mode 100644
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diff --git a/assays/MetabolomicAnalysis/protocols/.gitkeep b/assays/MetabolomicAnalysis/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md b/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md
new file mode 100644
index 0000000..0ac5db9
--- /dev/null
+++ b/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md
@@ -0,0 +1,4 @@
+## Metabolomic Analysis
+
+Metabolites were extracted from individual isolates (n = 198) grown on the same agar medium used in mBA experiments with two organic solvents with different polarity, ethyl acetate and methanol, to capture greater small molecule diversity. Each bacterial strain was grown separately on 25% TSA plates (25% BBLTM TrypticaseTM Soy, BD with 1.8% Bacto-Agar; BD, Germany). After seven days of incubation at 25 °C, three to four agar plugs were taken from the periphery and inside of the bacterial colony. Agar plugs were crushed and washed with 500 μl water followed by extraction in 500 μl ethyl acetate and methanol. Between each extraction step, samples were vortexed for 30–45 s. After each extraction, the solvents were evaporated, and the residue was redissolved in 500 μl LC-MS-grade methanol and filtered through a 0.2-μm membrane into HPLC vials. Solvents for blanks (uninoculated medium) were extracted according to the same protocol. The extraction protocol was also used to analyse the inhibition zones upon inter-bacterial interactions. To this end, a bacterial lawn of a sensitive target strain, either R472D3, R480 or R553, was prepared as top agar and the antibiotic producer strains (R63, R68, R71, R342, R401, R562, R569, R690, R920 and R1310, respectively) were inoculated on top.
+The agar plugs were taken from the zone of inhibition and inside of the antibiotic-producing colony. In total, 20 interactions were analysed. All samples were analysed by HPLC-MS/MS on a micrOTOF-Q mass spectrometer (Bruker) with ESI-source coupled with a HPLC Dionex Ultimate 3000 (Thermo Scientific, Germany) using a Zorbax Eclipse Plus C18 1.8 μm column, 2.1×50 mm (Agilent). The column temperature was 45 °C. MS data were acquired over a range 100–3000 m/z in positive mode. Auto MS/MS fragmentation was achieved with rising collision energy (35–50 keV over a gradient from 500–2000 m/z) with a frequency of 4 Hz for all ions over a threshold of 100. uHPLC began with 90% H2O containing 0.1% acetic acid. The gradient started after 0.5 min to 100% acetonitrile (0.1% acetic acid) in 4 min. Two microliters sample solution were injected to a flow of 0.8 ml/min. All MS/MS data were converted to ‘.mzxml’ format and transferred to the GNPS server (gnps.ucsd.edu) (4). Molecular networking was performed based on the GNPS data analysis workflow using the spectral clustering algorithm (5). The data was filtered by removing all MS/MS peaks within +/- 17 Da of the precursor m/z. MS/MS spectra were window-filtered by choosing only the top 6 peaks in the +/- 50 Da window throughout the spectrum. The data was then clustered with MS-Cluster with a parent mass tolerance of 0.02 Da and a MS/MS fragment ion tolerance of 0.02 Da to create consensus spectra. Consensus spectra that contained less than two spectra were discarded. Networks were then created from the single cultivation and from the competition experiments. Edges were filtered to have a cosine score above 0.5 (0.6 for interaction network) and more than four matched peaks. Further edges between two nodes were kept in the network only if each of the nodes appeared in each other's respective top ten most similar nodes. Sample attributes were assigned to the data files (strain, genus, family, order, class phylum extraction solvent). For the network analysis, all nodes that contained ions from the blank medium were removed. The network was visualized using Cytoscape 3.5.1.
\ No newline at end of file
diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
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delta 3422
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index 7cb4cae0c5cf802a64f2339b1fdc28538899b8b3..305ebbc405a57b8d78bfeb275ecfb564e85eafc6 100644
GIT binary patch
delta 647
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delta 647
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 4494a085727022919adf6ba46033d9b119d9bc05..3ecab80be028f406a045cbf75e94115fbf46e6e1 100644
GIT binary patch
delta 790
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delta 790
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
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-- 
GitLab


From b8bbd4025c43e93eb02fe063c8a74e93dc89e6f5 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 6 Feb 2025 14:30:48 +0100
Subject: [PATCH 12/35] metabolimics assay test

---
 .../dataset/Metabolomics.md                   |   3 +++
 assays/MetabolomicAnalysis/isa.assay.xlsx     | Bin 8680 -> 10547 bytes
 .../isa.assay.xlsx                            | Bin 11666 -> 11665 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7875 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12307 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15938 bytes
 6 files changed, 3 insertions(+)

diff --git a/assays/MetabolomicAnalysis/dataset/Metabolomics.md b/assays/MetabolomicAnalysis/dataset/Metabolomics.md
index e69de29..87595d1 100644
--- a/assays/MetabolomicAnalysis/dataset/Metabolomics.md
+++ b/assays/MetabolomicAnalysis/dataset/Metabolomics.md
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diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 312ee8adba84ba51e4d90f15983ad73fff565f15..0f385f7c66181b40c067c203e03f5d51f2cc6851 100644
GIT binary patch
delta 836
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delta 807
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index 305ebbc405a57b8d78bfeb275ecfb564e85eafc6..fe3fbd960299015f3bc97cb36f9ff682006cb4ae 100644
GIT binary patch
delta 661
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delta 662
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CSP0bs

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 3ecab80be028f406a045cbf75e94115fbf46e6e1..3cfa4b5e1e674de8b26132f233ec42cdf7e1a633 100644
GIT binary patch
delta 803
zcmbP|FgbxYz?+$ci-CcGgW-I(+eY4HjLblK@-{|A5WV>pBR@po4U-&Lf|pqZEMdZ|
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delta 804
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index e9fd0dca9c6296a9789b665e64ea1622c299587b..1dab431c8b16177ee9d95b92fef8c54b17a66f02 100644
GIT binary patch
delta 903
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DihgCt

delta 923
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-- 
GitLab


From b108a76651a635824ee7e38ad491e1190ce39ff0 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 6 Feb 2025 14:57:56 +0100
Subject: [PATCH 13/35] metabolomics assay

---
 assays/MetabolomicAnalysis/isa.assay.xlsx     | Bin 10547 -> 10742 bytes
 .../isa.assay.xlsx                            | Bin 11665 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12307 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15938 -> 15939 bytes
 5 files changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/MetabolomicAnalysis/isa.assay.xlsx b/assays/MetabolomicAnalysis/isa.assay.xlsx
index b90e1af038695d16e9691b0283b54fc1457dac5d..f9c134cac112875e9feb7fb8879a86c6e50a9ad0 100644
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diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 0f385f7c66181b40c067c203e03f5d51f2cc6851..d551349c89371bbe071c25f516a530e9371ea985 100644
GIT binary patch
delta 764
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delta 762
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index fe3fbd960299015f3bc97cb36f9ff682006cb4ae..7f49926976893ca51466264cda2e2a43bcd25bef 100644
GIT binary patch
delta 632
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D9c}(x

delta 631
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 3cfa4b5e1e674de8b26132f233ec42cdf7e1a633..370d6099ad8a743fa399232e8f79d7ad2c409dae 100644
GIT binary patch
delta 764
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delta 774
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 1dab431c8b16177ee9d95b92fef8c54b17a66f02..22e25ca925579e857d6a956701dca77f9b616d98 100644
GIT binary patch
delta 829
zcmX?9bGU{lz?+$ci-CcGgTXj|BhLm#Cgc3c`xupg)aFNw0*uPW`EF4_X*gg8vMO@)
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delta 839
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znzn7R?54^0Ek!3QSsR0tg<FF)&9pWH@h(Dm!Zu*RPzdF3Av1ZijU-6^oDJB8{I-@L
k9!Mh17A(<dYlD<(j@g1u`)4b|xOB3#of2E3IVf8I0QQGd3IG5A

-- 
GitLab


From f30198a359c039bd032f07f14b9df2b066519b34 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 6 Feb 2025 16:01:02 +0100
Subject: [PATCH 14/35] metabolomics update and DAPGdetection assay

---
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 11885 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/DAPGandPyoverdineDetection.md   |   3 +++
 assays/MetabolomicAnalysis/isa.assay.xlsx     | Bin 10742 -> 10742 bytes
 .../isa.assay.xlsx                            | Bin 11666 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15939 bytes
 10 files changed, 3 insertions(+)
 create mode 100644 assays/DetectionOfR401DAPGandPyoverdine/README.md
 create mode 100644 assays/DetectionOfR401DAPGandPyoverdine/dataset/.gitkeep
 create mode 100644 assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
 create mode 100644 assays/DetectionOfR401DAPGandPyoverdine/protocols/.gitkeep
 create mode 100644 assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md

diff --git a/assays/DetectionOfR401DAPGandPyoverdine/README.md b/assays/DetectionOfR401DAPGandPyoverdine/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/DetectionOfR401DAPGandPyoverdine/dataset/.gitkeep b/assays/DetectionOfR401DAPGandPyoverdine/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
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diff --git a/assays/DetectionOfR401DAPGandPyoverdine/protocols/.gitkeep b/assays/DetectionOfR401DAPGandPyoverdine/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md b/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md
new file mode 100644
index 0000000..b81ba3a
--- /dev/null
+++ b/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md
@@ -0,0 +1,3 @@
+## Detection of R401 DAPG and pyoverdine
+
+For the detection of iron-chelating compounds, a R401 preculture was grown over night in 5 ml LB medium at 30 °C and 200 rpm. Before inoculation of the main culture, cells were washed twice with the main culture medium to remove potential traces of iron from the medium. Erlenmeyer flasks containing 20 ml of modified MM63 (KH2PO4 13.61 g/L, KOH 4.21 g/L, (NH4)SO4 1.98 g/L, MgSO4*7 H2O 0.25 g/L, NaCl 0.5g/L, Glucose * H2O 5,00 g/L, pH 7.1 KOH/HCl) with or without addition of 0.0011 g/L FeSO4 * 7 H2O were inoculated with 100 μl of preculture and cultivated for 6 days at 30 °C and 200 rpm. Every second day, 0.5 ml sample were taken, cells were removed by centrifugation and 5 μl of  supernatant were analysed on a Bruker microTOFq-II high-resolution mass spectrometer coupled to an Agilent 1290 UPLC system with an Acquity UPLC BEH C-18 reverse phase column, run in a gradient of MeCN/H2O + 0.1% formic acid. Higher accuracy measurements were performed on a maXis-II qTOF, coupled to an identical LC setup as described earlier.
\ No newline at end of file
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-- 
GitLab


From 2fa3ff0d06612518570097dd3cbb61fafc74d5ba Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 6 Feb 2025 16:25:47 +0100
Subject: [PATCH 15/35] renamed Metabolomics assay, antiSMASH v6.0 assay added

---
 .gitattributes                                |   1 +
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../dataset/Table S2.xlsx                     |   3 +++
 .../isa.assay.xlsx                            | Bin 0 -> 9258 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/BGCAntiSMASH.md                 |   3 +++
 .../isa.assay.xlsx                            | Bin 11885 -> 11885 bytes
 assays/Metabolomics Analysis/README.md        |   0
 assays/Metabolomics Analysis/dataset/.gitkeep |   0
 .../dataset/Metabolomics.md                   |   0
 .../isa.assay.xlsx                            | Bin 10742 -> 10746 bytes
 .../Metabolomics Analysis/protocols/.gitkeep  |   0
 .../protocols/MetabolomicAnalysis.md          |   0
 .../isa.assay.xlsx                            | Bin 11666 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15939 bytes
 18 files changed, 7 insertions(+)
 rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/README.md (100%)
 rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/dataset/.gitkeep (100%)
 create mode 100644 assays/BGC prediction using antiSMASH/dataset/Table S2.xlsx
 create mode 100644 assays/BGC prediction using antiSMASH/isa.assay.xlsx
 rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/protocols/.gitkeep (100%)
 create mode 100644 assays/BGC prediction using antiSMASH/protocols/BGCAntiSMASH.md
 create mode 100644 assays/Metabolomics Analysis/README.md
 create mode 100644 assays/Metabolomics Analysis/dataset/.gitkeep
 rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/dataset/Metabolomics.md (100%)
 rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/isa.assay.xlsx (63%)
 create mode 100644 assays/Metabolomics Analysis/protocols/.gitkeep
 rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/protocols/MetabolomicAnalysis.md (100%)

diff --git a/.gitattributes b/.gitattributes
index fdeb834..6608dce 100644
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@@ -3,3 +3,4 @@
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 assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:space:]]interactions/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
+assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/MetabolomicAnalysis/README.md b/assays/BGC prediction using antiSMASH/README.md
similarity index 100%
rename from assays/MetabolomicAnalysis/README.md
rename to assays/BGC prediction using antiSMASH/README.md
diff --git a/assays/MetabolomicAnalysis/dataset/.gitkeep b/assays/BGC prediction using antiSMASH/dataset/.gitkeep
similarity index 100%
rename from assays/MetabolomicAnalysis/dataset/.gitkeep
rename to assays/BGC prediction using antiSMASH/dataset/.gitkeep
diff --git a/assays/BGC prediction using antiSMASH/dataset/Table S2.xlsx b/assays/BGC prediction using antiSMASH/dataset/Table S2.xlsx
new file mode 100644
index 0000000..3efd1aa
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diff --git a/assays/MetabolomicAnalysis/protocols/.gitkeep b/assays/BGC prediction using antiSMASH/protocols/.gitkeep
similarity index 100%
rename from assays/MetabolomicAnalysis/protocols/.gitkeep
rename to assays/BGC prediction using antiSMASH/protocols/.gitkeep
diff --git a/assays/BGC prediction using antiSMASH/protocols/BGCAntiSMASH.md b/assays/BGC prediction using antiSMASH/protocols/BGCAntiSMASH.md
new file mode 100644
index 0000000..567d654
--- /dev/null
+++ b/assays/BGC prediction using antiSMASH/protocols/BGCAntiSMASH.md	
@@ -0,0 +1,3 @@
+## BGC prediction using antiSMASH
+
+Bacterial genomes were downloaded from “www.at-sphere.com” or NCBI and https://antismash.secondarymetabolites.org/ version 6.0. Output data from antiSMASH analysis are listed as BGC classes and predicted BGCs for each genome in **Table S2**. Only high-quality genomes, as assessed by CheckM with ≥90% completeness and ≤5% contamination ratio were used for the analysis. For R401, the PacBio-sequenced high-quality genome was used for BGC prediction using antiSMASH.
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delta 2266
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diff --git a/assays/Metabolomics Analysis/protocols/.gitkeep b/assays/Metabolomics Analysis/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md b/assays/Metabolomics Analysis/protocols/MetabolomicAnalysis.md
similarity index 100%
rename from assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md
rename to assays/Metabolomics Analysis/protocols/MetabolomicAnalysis.md
diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 7b1eac32a44c5ac4a1db49f25ca84aa1e1c90859..6a295196d654f6892e0baeae865a72e6504fcebf 100644
GIT binary patch
delta 757
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delta 757
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index 2a088ed43ac0fcf0a890e7ad6c02ebbf8b78ca1c..86ae0c4a13fcd1a7383820cd9a06606ceeb7774b 100644
GIT binary patch
delta 647
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delta 647
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 96ce5007ccd6dc34aa7aa4bac652723ef03e3027..8133fff8be918e6c123098044d41f0c372ece6db 100644
GIT binary patch
delta 790
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delta 790
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-- 
GitLab


From 2201ca41668fd197bf4acd33cce291b069f89bf8 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Feb 2025 16:39:04 +0100
Subject: [PATCH 16/35] Mutant generation 1st part

---
 .../isa.assay.xlsx                            | Bin 9258 -> 9257 bytes
 .../isa.assay.xlsx                            | Bin 11885 -> 11884 bytes
 assays/Metabolomics Analysis/isa.assay.xlsx   | Bin 10746 -> 10745 bytes
 assays/Mutant Generation/README.md            |   0
 assays/Mutant Generation/dataset/.gitkeep     |   0
 .../Mutant Generation/dataset/Table S4.xlsx   |   3 +++
 assays/Mutant Generation/isa.assay.xlsx       | Bin 0 -> 8640 bytes
 assays/Mutant Generation/protocols/.gitkeep   |   0
 .../GenerationOfpK18mobsaB-derivedPlasmid.md  |   5 +++++
 .../protocols/MutantGeneration.md             |   6 ++++++
 .../isa.assay.xlsx                            | Bin 11666 -> 11665 bytes
 isa.investigation.xlsx                        | Bin 7876 -> 7875 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15939 bytes
 14 files changed, 14 insertions(+)
 create mode 100644 assays/Mutant Generation/README.md
 create mode 100644 assays/Mutant Generation/dataset/.gitkeep
 create mode 100644 assays/Mutant Generation/dataset/Table S4.xlsx
 create mode 100644 assays/Mutant Generation/isa.assay.xlsx
 create mode 100644 assays/Mutant Generation/protocols/.gitkeep
 create mode 100644 assays/Mutant Generation/protocols/GenerationOfpK18mobsaB-derivedPlasmid.md
 create mode 100644 assays/Mutant Generation/protocols/MutantGeneration.md

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diff --git a/assays/Mutant Generation/protocols/.gitkeep b/assays/Mutant Generation/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mutant Generation/protocols/GenerationOfpK18mobsaB-derivedPlasmid.md b/assays/Mutant Generation/protocols/GenerationOfpK18mobsaB-derivedPlasmid.md
new file mode 100644
index 0000000..00ed2cd
--- /dev/null
+++ b/assays/Mutant Generation/protocols/GenerationOfpK18mobsaB-derivedPlasmid.md	
@@ -0,0 +1,5 @@
+## Generation of pK18mobsaB-derived plasmid containing flanking regions of the gene of interest.
+
+Primers were designed to amplify a 750-bp DNA sequence (i.e., flanking region) directly upstream and downstream of the target region, sharing terminal sequence overlaps to the linearized pK18mobsacB vector and the other respective flanking region using Geneious Prime. R401 genomic DNA was isolated from 6 μl dense R401 culture in 10 μl of buffer I (pH 12) containing 25 mM NaOH, 0.2 mM EDTA at 95 °C for 30 min, before the pH was readjusted using 10 μl of buffer II (pH 7.5) containing 40 mM Tris-HCl. The R401 genomic DNA was used for amplification of the flanking regions through PCR using the respective flanking region-specific primer combinations **(Table S4)**. PCR was conducted with 0.2 μl Phusion Hot Start High-Fidelity DNA polymerase (New England Biolabs) in 20-μl reactions containing 4 μl 5x Phusion HF buffer (New England Biolabs), 0.4 μl 10 mM dNTPs, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 2 μl of R401 genomic DNA as template, filled up to 20 μ1 with nuclease-free water. The tubes were placed into a preheated (98 °C) thermal cycler set at the following program: 98 °C for 30 s, 35 cycles of 98°C for 7 s, 60°C for 20 s, 72°C for 15 s, then a final extension at 72°C for 7 min.
+Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye (6x; New England Biolabs), loaded on 1% agarose gels containing 0.05% EtBr, and run at 110 mV. After confirmation of successful amplification, the PCR product was purified using AMPure XP (Beckman-Coulter) and subsequently quantified using Nanodrop (Thermo Fisher Scientific). Plasmid purification was performed on an *E. coli* culture containing plasmid pK18mobsacB using the QIAprep Spin Miniprep Kit for plasmid DNA purification (QIAGEN) following the manufacturer's instructions. The pkl8mobsacB vector was then amplified and linearized through PCR using the PKSF and PKSR primers
+361 **(Table S4)**. PCR was conducted with 0.2 μl Phusion Hot Start High-Fidelity DNA polymerase (New England Biolabs) in 20-μl reactions, largely as described above with 1 μl 0.1 ng/μl pkl8mobsac as a template. Annealing temperature was decreased to 55 °C and extension time increased to 150 s for each cycle. Template DNA was digested by DpnI (New England Biolabs) in 50-μl reactions containing 1 μl DpnI, 1 μg DNA, 5 μl Cutsmart buffer (New England Biolabs) and filled up to 50 μl with nuclease-free water. The tubes were then incubated at 37 °C for 15 min followed by heat inactivation at 80 °C for 20 min. Five microliters of the DpnI-digested plasmid were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification of amplification and digestion, the remaining sample was purified using AMPure XP and subsequently quantified using Nanodrop. Linearized pK18mobsacB and both flanking regions were mixed in a molar ratio of 1:3:3 into a 10-μl total volume, added to 10 μl 2X Gibson Assembly® Master Mix (New England Biolabs) and incubated at 50 °C for 1 h.
diff --git a/assays/Mutant Generation/protocols/MutantGeneration.md b/assays/Mutant Generation/protocols/MutantGeneration.md
new file mode 100644
index 0000000..6a3d7f4
--- /dev/null
+++ b/assays/Mutant Generation/protocols/MutantGeneration.md	
@@ -0,0 +1,6 @@
+## Mutant Generation
+
+Marker-free knockouts in R401 were generated through homologous recombination using the cloning vector pK18mobsacB (GenBank accession: FJ437239), which encodes the *kanR* and *sacB* genes conferring resistance to kanamycin and susceptibility to sucrose, respectively. In this method, upstream and downstream sequences of the gene to be deleted are integrated into the pKl8mobsacB suicide plasmid by Gibson assembly. The resulting plasmid is transformed into BW29427 *E. coli* cells and subsequently conjugated into R401. The plasmid is then integrated into the chromosome by homologous recombination and deletion mutants are generated by a second sucrose counter-selection-mediated homologous recombination event. The protocol is adapted from (14).
+
+
+14. B. H. Kvitko, A. Collmer, Construction of Pseudomonas syringae pv. tomato DC3000 mutant and polymutant strains. *Methods Mol Biol* 712, 109–128 (2011).
\ No newline at end of file
diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 6a295196d654f6892e0baeae865a72e6504fcebf..250723be73887f05e87494613c1baa20b494819c 100644
GIT binary patch
delta 837
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delta 816
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index 86ae0c4a13fcd1a7383820cd9a06606ceeb7774b..f1b2aad87420abbc5ca88cfd23732ab62481cb87 100644
GIT binary patch
delta 691
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delta 691
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index 8133fff8be918e6c123098044d41f0c372ece6db..b76d0b9db4cb646638c22039b431ed668870482b 100644
GIT binary patch
delta 818
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delta 818
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diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 4ae933e454ea6eda4b8c0ffa618c2175619a86c4..007d8a5772a7fccd375648ac4f399846ec4da17c 100644
GIT binary patch
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delta 918
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-- 
GitLab


From daed91c5305ac9e6063a8dff04bff8692b86ebd3 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 21 Feb 2025 12:50:24 +0100
Subject: [PATCH 17/35] assay for mutant and plasmid generation

---
 .../isa.assay.xlsx                            | Bin 9257 -> 9257 bytes
 .../isa.assay.xlsx                            | Bin 11884 -> 11884 bytes
 assays/Metabolomics Analysis/isa.assay.xlsx   | Bin 10745 -> 10745 bytes
 assays/Mutant Generation/isa.assay.xlsx       | Bin 8640 -> 12624 bytes
 .../isa.assay.xlsx                            | Bin 11665 -> 11665 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7875 bytes
 .../isa.study.xlsx                            | Bin 12308 -> 12307 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15939 -> 15938 bytes
 8 files changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/BGC prediction using antiSMASH/isa.assay.xlsx b/assays/BGC prediction using antiSMASH/isa.assay.xlsx
index 971e236c3280b2ec6ea0d46cbedbfee04df97b4c..ab438f400679e35e0de49967e3a6af6f9a499bb5 100644
GIT binary patch
delta 758
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delta 758
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diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
index 3c062ac3178f27a26b90e39d142da28830457022..69890d3be81d994fb8a52d2a22db9d79c3b0b62c 100644
GIT binary patch
delta 818
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diff --git a/assays/Metabolomics Analysis/isa.assay.xlsx b/assays/Metabolomics Analysis/isa.assay.xlsx
index 8985d769d2e8f6d8a3f4775601e2e34b20a7c3b7..3419fe3fdb9b0a78280c93f59985e6a6327c7c18 100644
GIT binary patch
delta 758
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diff --git a/assays/Mutant Generation/isa.assay.xlsx b/assays/Mutant Generation/isa.assay.xlsx
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GIT binary patch
delta 8030
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diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 250723be73887f05e87494613c1baa20b494819c..5c996e6d84d020f9c80090083ac9f00e1d7d1763 100644
GIT binary patch
delta 800
zcmbOjJu#X$z?+$ci-CcGgF!1UbR+LFMrI&Ac^ji5h~9jQksl)PhDi=A!ON@ymM~$~
zfk-5?Xo3a$S++BQ8J_Gv!HmhST>4-w<y`N<5}DkOArk(4AHV`m{4<%sD((rXf(6)w
z&oJ=;t&L*W)LV6liGg7e&tySi(am2)A3}8AlmOelNpcq(Si@fVc@T*>C5W?HmCi77
zfJ|^dxQBCcq>4hl-(drRJ)cD<zF#?6M8G9ei}6*bmxoB>4$11<v(&bw=6inrZqdK2
z_eJOJbLZYv$e(-GzKNsmfYr*YJx3;Yuq@ai7L=RzBzG&vdyfYbG!JsEO>oO9NSPO(
zdS^*Ybj8M74X&JeAKXqYy>sS-*u^DNtoF4|+SwVd;lFOHu4Gk)`IUNueaX@bJVowa
z5}Lr-)??PTP*TP-U-L&o=C^Gbye!KD95}gTd({nXGX-;A>WOVRGjr3F2<aUfwv(EF
zJ$Rz9W>r>@Z}iId_RHi?KfhjfC16!rsEo%v&Ur=LJX_D+zvB1OEA@-c^$!{w8}|5q
zU)X==-)m`$vX_iMA{ritNqs4Qm31QU(EMcf(xqz)=E*L67Q~adIQMR#`V+^4GJ1b^
zUKcvzT>I*){?xC#-=;AL{+PSuE?fVZ-(NmvZdIrhQZ-MU(&D+N>+zRgcD{?wFMA&N
z_vD5*@;|pfzPMn1{ulkv@hFLB^F!4wkTfwv-3}bDH`NWnG_Qskn0C|91Jg5gq$f|(
zkOZ^WX}E&vZyJ_h+DOwGOjkhZeVX<l{v<6wuyCLjM0z@u|3V8aJy}cJ6fB>l4dE|=
n(jcoCbs$#B>Oh<or(*$DJ5$F5OkdEkMx+^GU30LYudWLK2w^pg

delta 800
zcmbOjJu#X$z?+$ci-CcGgW+UN$VT2}jLblK@-{|A5WV>pBR@po4U-&Lf|pqZEMdZ|
z1CdB((F6<hvutMqGd$UUf*F%tx%9zW%DLWyB{I1mLnQq9K7a+B_-8VMRooL&1q-kV
zpJCzyS{uc#skiD969dB{p2>p3qMN^pK7{DJDFL>9ljJTou!g<z^B@v&N)TtYDxG2E
z0GY5S%0Xaqq>4g)-XQ~lJ>RvJ?l(_X&=P4?*ebeQDO4+T^}U#CPQA|Do8Hd<_MK~H
z&{}@4tSs&L?}BYMVW}UOuX(N&Q<>+Y!f<r!gjEp}wx89SGx>mGaMRSNgH0w6P8jV!
zy~A_owhu{L8M}pI9`sJ}-WfSTc9ExMX$`w*vGDqUd3Um+4ZmCvyHankFP-&*O4qwg
z))OqwZqbJ?^z@}{5UJT>!X915))cI<kg2ur*yI`WwsLGMGUv8><`n6f!*gfKyc2=7
zB}F3FN^MszpL^xI{N?_ypR>)gG(vZ4@u{3=@!!eXcI#Pw*7}mQMpYBDE2l&_-CNpz
z!TsHbue>t9zi`y(94OT0{bl_%>q+RL`N{mgYu6Uc+w1gpsf5$Q+{?k~PaIFmtoyrj
zyU-Em;&)&5r}FN8o6aEkWA2i_Z2o6{fBBfXS*cP;b+dujA(dN4HkVlWKmM71`*Y~u
zn;WF$er|t!GGWI1SMksGqa>cq4^_87(!>mPJ8-<-R5t|Eyc%X;+D$_bOwZJjo;*oI
z63kww;R>d|X;^}3BTZ*8T>+){Y1)JMleGN6!hu>4>FH4Z3oWqpWG!t|uzZp>guetz
mgRElIfmkK0194WIjs;llOdS(2eL=?>k!FN-&B21cx-I}Nrc$o}

diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index f1b2aad87420abbc5ca88cfd23732ab62481cb87..ba6e39478168c92e5be36ba5756b6f5079d10710 100644
GIT binary patch
delta 676
zcmX?Xd)SsYz?+$ci-CcGgF!1UbR%y&BQubmJe^SyL~q{B$PW=X%OnSuc*mpymJnr@
zgGiXMXo3ZjSUxj@84(<^EMTP-JP@Vxd3LdZWnzTqF@a_Nib7;m#5OZ>0Ii8~Ke&f;
z@_liIdcVU40((A-PJF*|vWS37rWWI?PA?CU#vPK?w`ZwsOU?KE{N18|S?`O^+vm=`
zsgOVStbG$l-2tnWS9^|3?qFH4Lo6sa?Md!dj`to9CTJeyTASdORgf|-KK0I$nCOa)
zw;EhI^**?rT6*Wq39*YyrdaK3owTzvT*H6eR$a-e4D&1X2K$nw7kG-?y(Bb&v#rOh
zZK0%$XTIi-gv@W-GI&{*2RLwY$@Z!n+GYyoywnrha%SeHDG|~;G;Aj||9bF5Va=+n
zAm8Yf@9me#pMHM5>`K6@v``t3d7Sf#xOujoy?@2;rB~_~o$DVoHa6_>{l2jO&cD~v
z7G*CPe?&Aq4wL#){;K{&;Gy}+?4?WB7R-}f_$-JgadGb5K=mh%2W9mB?z}E^#JTp>
zSN*A9cfU<z5d1NB$z8VoGrzxl%-pI_DWqziIHkpNPuJrwzwCS$onQ7m@bAeDZ{&Y&
ze|&Ml{QNKapW{)YI;yMY+3EyF28J3Y1_pj$0;tH*FW+1vSq_QTZ&G&Ps4|x}1k>5l
zW?*`Wv>uo~Eh|0wgR~@=%_HLqrbA>b!Sn<fXE1$D#vV+A)Edh|)cVUp%$*=>0hKcW
T(@b*Kh?L+eXATyumvaFCYn=`;

delta 676
zcmX?Xd)SsYz?+$ci-CcGgW+UN$VT3FMrI&Ac{-yah~B)Lksl&(mPrmQ@s3FaEFsD)
z2azyi(F6-5v3zC*Ga@)-S-?swcpysW^Xy^+%ftxJV*<<k6@|#Ch;3%%09q5ZC(1!!
z@_liI`n*F10(-t|E8TCNte_>*s<2gbxl*WB=<0hh)tq{rxi`I?|Lr^1%%HXWURhb%
z@!tj8Y{F7MFkkarE2c8fMTOz$)(NX3CTu^eHD~ex#o(r?Q3sn$9-J`Re|m@K&TSu(
zwla1L#XRVp;=MC+g6tws&C(in(PH8C0rT!;MH_y(Aa<qRU|%}x1(mLMnXD&RoZX@i
zU+C#e*&tH0#e_Y&jIAkHV<A&(-?7Ov=56KJR%Fg?^~@>KGl%ESlzArtYfFkmu9e!Z
zTt4^8clpcxUq5G?XK94)*5Xq+&*Hz6we8lk{H*mQYmKTVW>-#$aJsj&{et_u4_|p@
zet+Sp(K%44&HKyxYyFeZL-UjQeb=rnn77yI?NSM+g}Il5)1Nq=lv($8=XRkZ&c*M(
z>QCj}{WhIJ@W<RGf7$%c{QmMWbF)&Vkm_awuR|)gj%+Tm@_+m@{r2b3zc)8X$^G2^
z_+-M2_pjog?MI2~sIHo4s}mR*7;2ap82Et+pdv@Vd~=OtIV4)YN!fv;%3RtIOlM1*
zf$1gEdSLprtn}m$(vn~{kBlpr4w110(-UNz!Spp5doT@BYb*;<>n{s2cY>@1RL%rU
SGs#&aQi7|TIash>&IJJTOCOa0

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index b76d0b9db4cb646638c22039b431ed668870482b..e6aa52d765bb890d581bcbb4eb1d33dd61ecada6 100644
GIT binary patch
delta 833
zcmbP|FgbxYz?+$ci-CcGgF!1UbR+LFMrI&Ac^ji5h~9jQksl)PhDi=A!ON@ymM~$~
zfk-5?Xo3a$S;|?!3{K9QOkkx?x%I)cIL~{C1n(z^#6SKIV1bVU{a`JVn?+T@0;@$=
zgH>#flwe~9>rIk|Sl21Niw&&AO=%v)kmo8889vn$jGREHM!6r{!x=aEzM6c!-(drR
zJ)cD<zF#?6M8G9ei}6*bmxoB>4$11<v(&bw=6inrZqdK2_eJOJbLZYv$e(-GzKNsm
zfYr*YJx3;Yuq@ai7L=RzBzG&vdyfYbG!JsEO>oO9NSPO(dS^*Ybj8M74X&JeAKXqY
zy>sS-*u^DNtoF4|+SwVd;lFOHu4Gk)`IUNueaX@bJVowa5}Lr-)??PTP*TP-U-L&o
z=C^Gbye!KD95}gTd({nXGX-;A>WOVRGjr3F2<aUfwv(EFJ$Rz9W>r>@Z}iId_RHi?
zKfhjfC16!rsEo%v&Ur=LJX_D+zvB1OEA@-c^$!{w8}|5qU)X==-)m`$vX_iMA{rit
zNqs4QRevJz(EMcf(xqz)=E*L67Q~adIQMR#`V+^4GJ1b^UKcvzT>I*){?xC#-=;AL
z{+PSuE?fVZ-(NmvZdIrhQZ-MU(&D+N>+zRgcD{?wFMA&N_vD5*@;|pfzPMn1{ulkv
z@hIsf>VKKd|5io@hCfUU4E(@URFR`!zPUzY4kT?zYuSNgF-FS}Oi$1<1Jft9^uV-?
zzVu{KZAmcOP}>zuS87{=>8;w%VEQ+dcGR&4M{{P?j3XN-E9i<&o~L6BR&hlKqEbrN
z56sWjwFlE{bxpzaQz&0u4<Zh-wM7qN>jFK93m@uPfYr+An}F$HeQTsdHC-R#?hE?T
RjFFSy=qs^tYXLpW0064sKNtW2

delta 833
zcmbQ7FeQOEz?+$ci-CcGgW+UN$VT2}jLblK@-{|A5WV>pBR@po4U-&Lf|pqZEMdZ|
z1CdB((F6<hvy`)d8JwIqnZQb)a_fU>ah~@O3Eoc-iGTbbzycoy`oUTzH;bx*1y+l$
z2CLW{DZ$1J)|(^^v942k7aLfKo6<apA<tDHGJL8h7&(DXjhc6~-amfweKq;|yh8>8
zd%kNc-EW?(pe5C+uvK)qQmEF7(EN3K8@!XEzg?QJfBk2LhEprI&z*boqurdd{i%X~
z8cKs!OLn>oayk_1F1dB?MAcS~<thayrX;k687P}t7@dD_lozy4cTZIAfkiCh70OS8
z^3qb;GlINi?>nBnb3%KHy7{eXJhoe+v+5=8Z`Qq_()BKr^8|~toAlueJbg>Hi_~l|
zVUOOz))cI<fTgwX*y0)UwsLGMG3SnY<`n6*hWE~tc_$oeONs=-LQPj4TYKfZ{N?_q
zr=zX2G(tCP@u{3=@!!eXcI#R8*6>|Hl6y~>+IZ<G<X^6Rp<Mps7h7L!6@#7FgB?@Z
z{?4_k|D^Ts{KocOm%=R0$1Qvo#FMx>_ikYN6UT!xdVhDC3mr+WefL#=>et<G(-;JQ
z%w2Mq&Hv2rA1^DnDpU%oPB!p5q!M*>a*2)a<M#8*p0ED3Iq|0br~KoS9qsR5-G8Q@
z5hcMy{V%im-^$3q@P~<kfghNRDsuEUS82?Jqzx%8J8&#UYZ-#+el0UFeL_nQOiSxa
zPZrUZ1hWmaUBPsPwk4R}qU{W(e?e&n9eZ#zXI9NPvT?G!uIS{sI>ulXmvta2C3XG4
z{48C2Fug|C6ih#X^40Vp;vid_^&qy+*Mqq5fu03et+c)gm=4mnMoLuE^das(uP@CQ
PHTku^5*v>e(60;t8Ch9{

diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 007d8a5772a7fccd375648ac4f399846ec4da17c..293a48d741bfac0803a331bcaa4f1e559d6bf046 100644
GIT binary patch
delta 933
zcmX?HbEt+lz?+$ci-CcGgF!1UbR+M2MrI&Ac|W5fh~E5|k)Kfo$cO@}fCFYAt0G4q
zrjr4rbFvt-3Rs6NvoXYoVirxXz<ie3ED#ya#bCzd$GrL=fyttL@4*u6{87xDAWIxC
zx6Ro!S(;B}a-YyKuvECP88cYz1hEfbfo5?xHn6}MSyiyW7ul0w6`Kzz_A~Q=Oi|7I
zvO^8%#x{Y;|7Art7pa;;j98@sap6UcU0@?N_vz1L0-L381d$0dI>QKdi2K1koN<%2
zP2}tS4jTyU`7Ao|{mRKA0xp?ajITPqJVY9INLJsTrM4|K-}Cc#i~eQ3FFJ3ZJNKqS
z{@k<nO&oOxtX5v_IWoC}Wx)=ypxm@4xm!8jdpww+d5~*uf?HNW%Dnj0J4<4sD>mM0
zaOKqd;C5>1oiiuIE-sm3wXb#3&dzWR|8-k+C95*buhbjtOO{^XDRTFc&;-u59<#QE
zk}{t8nm-aUzirFlWmz8Jz{w@st8QqUDVXz8Pi)JXnVY6WNbk_Foz(p6!4riwtFnT8
zqgTGSUnYP0`Sr3Z0jttNWjy9_&MV^P*?RW=6~C8Wsb6%if6&<2u*diN!u~t|UQ1h)
zy=43m(eOA->Pz{n`V)bN<|nh4E?rwNPj=z6AfCj<xpxEApEw?r(fhmey3i5l+E-uo
zr+(f2HjP2>$J`}%+4|4?{_-(%t3suas(Ip+7SBChkH7q~^Idd)+4I1^CpWy2|GE9~
z#Rc>8zvzFChovBJ+KW;=7Qelpk%7Sq7<c@TBv!upgy}L!aw;^p1IN`Sb3-uw%-jr2
z%UkGy=>Q88Fx_t93Z~CnSb}M8OJ^`02&Jc6+JpHwE&af>mK8+4zzV`YXypt}0%_Y8
z%Wj%{-%@n4lC?2dWrQ_E=PYYeF#i&iFJc1`4};PH7SfY9+em`doVS6vTENx<%m>M(
g*+S%+Y^{+J+c8^+fBxG_GcKJhZKuSRXb#G80Ge4@`v3p{

delta 923
zcmX?9bGU{#z?+$ci-CcGgW+UN$VT4vjLblK@_t4|5WV>^BR``GkP!t`0SC-LRz;3J
zOeX_K=VUQv6|fFlW@CsE#Vnd&f%z=6Ss*f;i@}V^k9qY$0+U7g-h(CB`J<RQL6$gP
zZkw}dvNWH{<UXNeV5x9nGiI>b31T0>0?p!XY+!*ivZ`Q#FR~}WDmEWb>}Tc!nWCEa
zWrrHjjco#x|I3PQE>bmx7_mwN;=+p>yTC?l?$e*g1U5_G2qF_^bcPY^ka<Vz{o^NV
zo5<Ja9WoHu^Ico%e)D7nEvZ(8t)k17LbX<e=C9k^;GGox?b3w(>pv?roLaek?%bOn
z?dF{APZj*rP#UyaveR9V)1gpz$*prIs<v`0S1C9#C80IUK-tv7==^)5yr6Zud!lj=
zEMgI_P<|SemzL6=5#%L%-|^&~6WUYM&2LTPvE34#RWEUWv+f0zu6LQ7Cs>@_qz_-<
z>07d0q-KK&d-N8zreKW)EUkUV7SEWsm1A3pId{}Er%10gymzL|JK<PcQX~)-YP#y!
z+AH7XFZVw^9c`VZ5xQB6Pvty||4!DnThFq$hVKfJ+<U^*#!E*b|8ngM<?<iD*!p6t
z80^Fz?3l{-cdk|aC#{F)H@5G(6lQTgZsD^ap2XF;cLUR(I3ASI`@7Rz=ty$yyRZ6F
zzwUmU#vu4(?vlG~{%3ywcv-nsp;Ab7vVqqjm8heWOKf}}x1V43eD$x*i8t*(<sYBy
zXn+6e{xkKAup|Udd{K(W;<xuRGB8*HBaa`F#x@@_T@Fc31?G0(xY}rL2&SK!n}KOL
z3q3IHZ(#za+bmqc^f?PlFwJG@45kC1^fXI*F#m?7ADGs(g2?AvLHGxtd^T$cKinFk
zex|i4Sp7vPU)TmB9tx%XEu<%JvXKO<IcEcLE5EG;m=BUmwS~wv+FBzev7@#SU;ML`
QW?VK|%1((b$sCl=0P<IKAOHXW

-- 
GitLab


From 42e3accd6ccd936e72f18ee35eca7db709c59e78 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 21 Feb 2025 16:09:44 +0100
Subject: [PATCH 18/35] transformation to E.coli

---
 .../isa.assay.xlsx                            | Bin 9257 -> 9257 bytes
 .../isa.assay.xlsx                            | Bin 11884 -> 11884 bytes
 assays/Metabolomics Analysis/isa.assay.xlsx   | Bin 10745 -> 10745 bytes
 assays/Mutant Generation/isa.assay.xlsx       | Bin 12624 -> 16547 bytes
 .../protocols/TransformationBW29427.md        |   3 +++
 .../isa.assay.xlsx                            | Bin 11665 -> 11665 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7875 bytes
 .../isa.study.xlsx                            | Bin 12307 -> 12307 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15938 -> 15938 bytes
 9 files changed, 3 insertions(+)
 create mode 100644 assays/Mutant Generation/protocols/TransformationBW29427.md

diff --git a/assays/BGC prediction using antiSMASH/isa.assay.xlsx b/assays/BGC prediction using antiSMASH/isa.assay.xlsx
index ab438f400679e35e0de49967e3a6af6f9a499bb5..b06b8354d92b038d824c16bc5ed5745d5632e109 100644
GIT binary patch
delta 719
zcmZ4KvC@Mlz?+$ci-CcGgF(7-BhO4mCh5w_%Ndn`)aKKS0$}c4CV7zPKPFX>s4BBM
zSk#Y23&gEr*~bK;t=JcV=*jmu4M3Ct*9VX&6Zb5z=saFE5ce4GQZP4H0BqD0f!%B%
z;Xv{EVBt@aU|~tAJ&YXEm7!4?H^SQ{-<4LV&pTuwu;;tB(*5Si3R)tq3R^{&D}`!>
zuD%yj&8gR!d(+$b-@bFr3|h<Ym6fF(|6Q=nCM@*>^EJ=4Vk+}oR2YtKov<om!uGRT
zb0!~93~rhlb+F0g!3m@Nr+0Yn-1Z@9D`U4%%!A%3-a8{F$S(5KEUjS|Ef!uMFz-%Q
zwBeTvVpr-7_NB94Q0aP?$$Emt*)96;g`U2Y4I(vLOxUB#*qVYh7BaQ=9h*F3-d2un
zMdsXA&zvGXb9nAdnRg<vwxmepTB+^I<#Vrmm%rTq^>em)mPY7qEk2d=EdD!L+ipF}
z&stxy)~IS?cIA`^r+Z7=FSx(^@Re8Q_ZN;Dodbp1yuYl!);|e7G(VZ&ckSAOd3&AS
zE|qXvn0q-m{fXmAnRS16ZWlV@T>S2<{#4%GZ_^nBf6QI-m(Bmo?=K%SH!D>Nscts#
zI;3*%$YzsS%QxRlzx_G%@68QTazD2}KAAA%{j2zA`;k*XXw>KGnuVo|3=BJ%7#R40
z>7pV>zkG9*Y!5ijIppm@k?SdM1fpu?%|X;Qd3_LNpe!?)SwRxSlv8j6QE3WRAZn3<
z3y69Fq4X3TK)eD)e-L#L!edr41M#etz;YmyW-5VA+M)!u@T-y~NZvr%6hx&f+aQw1
MYGtq^o+!Hl0J2aPdjJ3c

delta 719
zcmZ4KvC@Mlz?+$ci-CcGgF!29BhO4mCat*1%Ndn`)aKKS0$}c4CV7zPKPFX>s4BBM
zSk#Y23&gEr*~bK;t=JcV=*jmu4M3Ct*9VX&6Zb5z=saFE5ce4GQZP4H0BqD0f!%B%
z;Xv{EVBt@aU|~tAJ&YV$aiLM}2lsGJzALRz?|0ZhV9#gKiSJiV77=jC)M9+q>E$8P
zxI?n~_AIq+srjCtzgzS#>wVFA``o!V74qkvwQu66J7Bf)YR{3$9V`oWhy~@QJ;~k5
z@!sRX1kHn7YZKhE3R339r`}l-6J4?KR)Z_2-Uqi+OYfXHA$D=e6svu$lXiB7Yxu9*
zsw-KQVSc6FU|+KI0#A{<mxLy8w)L2`EtHh;%-8&pkoj#}1~1F<00&Mk*<N)++f2cn
zmwIAb&dl62B|>_KhV7*0Uk{!rtXY*6<Qu*6z5O!z)6cJ$T?tr~7AoU0k8@rTH_z6y
z_pkW9^h*7rbNz$H#)dt<-xv1Z`S)7dqU<H(kBElHVNzepU)7%oJTyO<y>#i?f_btF
zp9S$GF3!ChsQ$$9pp4$%o!5nqIM=@Vsz3GX?zd?Sf<NXixy#mn=J%J6nOhYqg;dQG
zr?hzP>3aO-m!0pT^UIzG{yn+jjr`B;k1sBmpZ`Vwb3Aei2#xw&U9+&1k%3_c69WT3
zFkMvS=$CJ<lI;P<IfuMGC~`gJjX+ebyg7*4Ca({o43uRiGb>1fm~skkASzA43PdeZ
zZ~;*-Ae5e>1Bh3k=ntX}LU_zdW+0xm5?Bso(o7|=Nn4b_7JgN-1j!pHn}VoxWgA5D
MSgj0p#1my#01!kO1poj5

diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
index 69890d3be81d994fb8a52d2a22db9d79c3b0b62c..e846156dd260e8768b142f4e0c7d8c94e6b7ff3c 100644
GIT binary patch
delta 763
zcmaD8^CpHTz?+$ci-CcGgF(7-BhPY1Ch5w_TN#yr)aILv0$}cICV7x353?#r)R<Wp
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delta 763
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diff --git a/assays/Metabolomics Analysis/isa.assay.xlsx b/assays/Metabolomics Analysis/isa.assay.xlsx
index 3419fe3fdb9b0a78280c93f59985e6a6327c7c18..c876c4ee3472462539cb9506cce354ad57473dfa 100644
GIT binary patch
delta 716
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delta 716
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PgGd|InqU`f(R2j>93L5T

diff --git a/assays/Mutant Generation/isa.assay.xlsx b/assays/Mutant Generation/isa.assay.xlsx
index dd96ac643f307d69ab419072c2fa5e8111b16456..0d4d9bfb148129fe76665a291dfab96bad264f67 100644
GIT binary patch
delta 8267
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diff --git a/assays/Mutant Generation/protocols/TransformationBW29427.md b/assays/Mutant Generation/protocols/TransformationBW29427.md
new file mode 100644
index 0000000..232c0db
--- /dev/null
+++ b/assays/Mutant Generation/protocols/TransformationBW29427.md	
@@ -0,0 +1,3 @@
+## Transformation into chemically competent E. coli BW29427 cells
+
+The vector was transformed into 50 μl chemically competent BW29427 *E. coli* cells according to the following heat shock protocol: 2 μl of the vector were gently mixed with 50 μl of competent cells, and the resulting mixture was incubated on ice for 30 min. The mixture was transferred to a water bath at 42 °C for 1 min and put back on ice for 2 min. Then, 1 ml of 50% TSB with 50 μg/ml diaminopimelic acid (DAP; Sigma-Aldrich) was added to the heat-shocked cells, the mixture was left to regenerate at 37 °C for 1 h and then plated on 50% TSA containing 25 μg/ml Kanamycin (Kan) and 50 μg/ml DAP. The plates were incubated at 37 °C overnight. Resulting colonies were validated by colony PCR using the M13F and M13R primers. Colony PCR was performed on at least four separate colonies with 0.4 μl DFS-Taq polymerase (BIORON) in 25 μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 10 mM MgC12, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM forward primer, 0.75 μl 10 μM reverse primer, a small fraction of a colony and filled up to 25 μl with nuclease-free water. The tubes were placed in a thermocycler set at the following  program: 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, then a final extension at 72 °C for 10 min. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml DAP and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol.
diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
index 5c996e6d84d020f9c80090083ac9f00e1d7d1763..c5fbcc9dda8928f7c4f7b86087023549c3be61da 100644
GIT binary patch
delta 757
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ko>2#Eyo?Um6|p*&AZ0UjOhMFn9UDaI5Y`2|%tzN10EVL`D*ylh

delta 757
zcmbOjJu#Xmz?+$ci-CcGgF!29BhPY1Cat*1TN#yr)aILv0$}cICV7x353?#r)R<Wp
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zZuf(GI44J{DAfBMHW1kJS#;w2m6JsTTr#y7Uv+wUh&1kytiC-<ZCh%-=jZPh{mXh^
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z37l;`W^D^4WjymWe<Wmn+m^x0vOK_nlS{T&-Ox5uFz2P7*p@RhH%*C<-l1VTsrlD~
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index ba6e39478168c92e5be36ba5756b6f5079d10710..b15f1a4b4e624c8fb1fa381bd79da504ac15d5de 100644
GIT binary patch
delta 647
zcmX?Xd)SsIz?+$ci-CcGgF(7-BTok-lXT_eX^cuhYV$5e0WkLrlRQZDEt4upRD@X`
zENaT41>z>Md|?LB;T&=-AXVi&U{&*YcC&$mqlM=)frS5vf`ygEwlH!?SB6Gq+z4-*
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z7kc_qHi*=0F=3A`V`~c5Sjg1ccWm;Ed0RQQ6`6BeJ#&im%;C8+W!{Ov+L9uXYo)d;
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z-(NUtbPg11^Zv5_TK^>U(EMb6-?eKC=IwQQyHvtyVeaMN^e2udW!C-Oxn1apbMd>c
z`crv#zfETl{4saQUpD_UzrTFU+^ke7q`KL_>yXO5Bb!ZTE#G`I{r2b3zc)8X$^G2^
z_+-M2_pjog?MIH>(5SAOXR8w!85nAq7#R40QC^XwU%t6UvK$<pU#09p@nj}#1fsH}
z%|X;+X?+lNN>*m_2Wd$VlUv3OL<P%OfvA2N7Z7z-#sNfulo`u{mHEknP3xDngh-fz
SC`LIOMBKZ|fo-Uha|HlKk^yl5

delta 647
zcmX?Xd)SsIz?+$ci-CcGgF!29BTok-lUCg1X^cuhYV$5e0WkLrlRQZDEt4upRD@X`
zENaT41>z>Md|?LB;T&=-AXVi&U{&*YcC&$mqlM=)frS5vf`ygEwlH#N#f3(>AKb$^
z`M$V9z29L2fjyr^C%#`fSwz4kQ;YFcr<aFF;||H{+q2ZRrRIBn{%+B~toKFd?Q`ee
zRLGxu*1n0O?ts<Gt35|1cd#tjAr_RI_9S;J$9s<l6EqKUtxa&tDoB|ZpL%CWOmxM@
zTMe$9dLP_QExmK*gxJL;Q>^y2PTJWSuHnCKtFB~KhWV9xgMG=;3p_>cUJ{zX+16v$
zwop>WGhg#ZLgu$^8N4jZ0~|QHWP8;OZ8HUPUh0W$IWu$9lnCh^8n%;~e?54jux3?O
zkZ<(L_x8)=Pd~q2b|qj{TBwZ2JkEJV+&o*)-oN7a(ku0g&h-x(8yoiceqY#s=ih5-
zi?WxDKO!0)he>@Ye^q}X@X-8Z_R^(m3+Bl#d=|u$xH$K2p!yTXgED%5cU~7d;#~Xc
ztNzrlyWgfU2>zJ6<StwPncrVNW^Pre6jC)$oYLaCr|a>TUv|EW&M$i&`1j<7H}XHX
zKfbtNe*PEz&+*8S8yeMB^K5kjBLhPX69WT3Fv=@(^vgHbNS1@6^Q)9SD4xuujX+eE
zv^j`cEUgcsPRYtl{va(0Vsgv4fv8{^D-hK$;{u|t$~b^1kTPRgurfbcuxb6WmJkV3
S5XC5GgNS=qIj{|Na;^Z^!vu2x

diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
index e6aa52d765bb890d581bcbb4eb1d33dd61ecada6..f79bce8581a85dd69170620fa7fb175e0bed438c 100644
GIT binary patch
delta 779
zcmbQ7FgbxIz?+$ci-CcGgF(7-BhPY1Ch5w_TN#yr)aILv0$}cICV7x(qO_<`d{Jsn
zu|Be%&Bn~SU{y&hS|Ck*EEOyunuGHe6G+z+ZUYb{#`6Iz%KI5C`j`JBi2Ffc0!Z=X
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zHHG@TLk0qSzH2MpZ=S56CDN*}Rdl&hs8;Cedok6VdY!p9y`BH<JJ-yhwftULS=#a6
z1>0=GQa><X^IR*YGS5YY;po;0t0E?BKdUuo@&U!*rm0Z}n@k>@Fxr25hv&|1ACk5*
zb_>Nk=$+!dGjf9LB2UfI8g|iQ;q?LY?qo$9ez_oarQTp)I_m|Mu6LQNCs>@_q7Pr_
z=}XxlQnST`J-UpoDOh76Q)}O`$us6{<=9qa&TaL~Dbh2C=gyRQCjx6ribSrJ+OAwa
z_sVzq%l%(JXPakfgznbjQ#sG#zmv7?*0cPq^(AYKswQSvPKj{3x3v9&`@0Wcd1Zco
z;i%C$P^iuO%ld2mlh8x+llgtut}U3i*Xiw238#g*mxI%vIG&VQ_jl)Zp(D=4@4o6!
z<=y=@ok8%&+$De6{LlRU@-cI>Ql*gUW&^K7D))|THkq}2^Ud_zpF{uN+#n_QbNl0y
z2{Ycmihs5rInqO;{+HSOZ)Id)_`}4&zz<A56*>Clo2xbEfRloxmOUu?qO^=aRG*eP
zh&ryN52B>>WhRSgOM;mC+HN4KT-ypnZPs=HQ9mJ+y^aHj*QnzUqAuxx<s@{$yi8rN
y+-hAjklbSkPgM^r2r|A|4{ZEAJ+Le8>sf-7N$Hz{s6c%iM4FkZ4|dr(eOCaq3n{_?

delta 779
zcmbQ7FgbxIz?+$ci-CcGgF!29BhPY1Cat*1TN#yr)aILv0$}cICV7x(qO_<`d{Jsn
zu|Be%&Bn~SU{y&hS|Ck*EEOyunuGHe6G+z+ZUYb{#`6Iz%KI5C`j`JBi2Ffc0!Z=X
zCQ&sIca`WGknH9N33g_XaH6y-h}$8(n+-&}D$NILex?Ez=2bn($e|S%8s&a)59j22
zY6|s!hYbYwd={Pfe&u8l0hdfI##fzQ9wLo9B&%=FQrniA@A>(=MgOwi7oE4yoqJOu
zf9_fPCXTuTRx7Xe9GTp~vS5c;P;T0j+^rn%JswQZJjk^+!7ZyGWnO&hoh32R6&r6g
zxN_=!a67g1&Y2Tp7ne-2+SfX1XJ@#E|GKTZl2sYzSLzM+B}*^x6uEmzXaZ+jk6GJ7
zNg2<4%^wMw-?nA&vMdj9;N+6+RX4QF6wG<4C${Cx%uQ1wq<3i8PHO)3;EBSTRars4
z(JSBEFOxt0{Ce4yfK_RsG9L3d=M{1DY(0Dbir-7G)Gs>MKWJ=h*yH<sVgH?fuca-@
zUNZiOXm}hZ^`-n({fWRs^OM<2m#!_CC%f=j5KrRb+`ED5PaF@*=>6S!UFe8&?W?c)
zQ@`$ho5mpcWA2i>Z2f0`fBBfXRiRQy)jV-Zi|3xM$6tQg`7S!Y?0MkdlN;X1|J?rg
z;)414U-Uo6BS(5@)c-P@|E-J+41bsy82Ew7ry@tcd~>zN9B@*Q)UpRfUzC;+i0aca
z2T{kh^g)!AzRYA1ZAlPQU)v2tm1|ppsLk3gAnGTCve$6{@fvmfLDVH3u$+W0n3t&w
zmRqfB29kRW;i>9@1wqC)>w%4*rw4Y$eLYK%GAVsi5EZCzgGe(|^}#MXr|${?-q<Vz

diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx
index 293a48d741bfac0803a331bcaa4f1e559d6bf046..36f4e75c4a78fa0fdeaa5b15460c17377974b9a2 100644
GIT binary patch
delta 822
zcmX?9bEt+Vz?+$ci-CcGgF(7-BhLm#Ch5w_`xupg)aFNw0*uPim7!5UX*gg8vMO@)
zVcHmg+9r!KtAf<qFq?q27qMu8xbs-%uz-a*mw@QWk9ZA0lnCDkkSH5}G&3j2aL3DS
zb2d$u=2My6Cv*%X6DDlV3{u!H_7TKw5_e|<aZk&tfw-S#Pl04N?^m1vrUO;Oz^Yej
zfZcFGV>d|k=3f2zOdwr)MquGkqqB@0Alou-gttxBGEu0{J7gfR=exGj{pQIES|Y6q
zTSb>Eg=&Saz86!?sn?l%)7$yqzH`kCTFdX1m8Bj3U9in2EcFBPHP5wTD)U@a7>;h8
zuqtB0_On`ZCLd4?Zkif(u*u}X38Vd|cX;mH_91C2W4BPugWf6LJ0mB^F7nhYtzj1}
z7G57P?@m^<;g<_ySLzM+rL$g8>3WyRdV<B-E&A|<p1za~A~jn~*rUtXnu0YJGPU*{
zn>=IQR*r2&=G<1#oFYAQc<xM@cOtO1q)6misqM<;bFX}tzuf=zbGCVwM(A!WK9%z<
z{ySOQZavG-T3@o(sA^(%<&+4gdrR9dxWD`Gl~?BX7mga81BKeWzpTGzJ_$WEKbhZm
z?b?EQd!61cm2g^^dpS7$iQ`F`b$@qm7dqlx{O+s%RNmch(-{PR%w6)A&Hv2rFCQ~E
zD^&`qZZ_~bq;l`bW|LXVH{VRZ{W<jS%?(m=Kes<VnK0x1tN3U8VW|U@s5Wmj-2+Y*
z`R4YZ7};QM1frgpn}aA>3w;pfXJHDWS}oi_)L9EF5XEWf0;2pO)Kp6c5bwIBKZw$>
z0?XxDfqDBOJXUKkFU%UOa)z}TNaY0xPsj!=7y_aEEMz8cvXKPIpS1xyi_g{)!~;pB
U+JYq-Y;6!p=7=rWxqoe40dbu*hyVZp

delta 822
zcmX?9bEt+Vz?+$ci-CcGgF!29BhLm#Cat*1`xupg)aFNw0*uO9aiLK_X*gg8vMO@)
zVcHmg+9r!KtAf<qFq?q27qMu8xbs-%uz-a*mw@QWk9ZA0lnCDkkSH5}G&3j2aL3DS
zb2d$u=2My6Cv*%X6DDlV3{u!H_7TKw5_e|<aZk&tfw-S#Pl04N?^m1vrUO;Oz^Yej
zfZcFGV>d|k=3f2zOdwr)MquGkqqB@0Aluvz?%|xQWuj2;ci2E+&u7tz?^jM15pc=W
zVtm!<<ss6zL$dnzEVXT^`JSJ@Tl6pMebIUQ+_^Uu^5>qlZ{nysV72mU&ymR;EDLss
z1?8qa$=%BF-s8ap&4XNP6Wp>2Qs%{{-dPe8U9s_2gDa=r2e(s8@0>Xyc5%rRt9`AL
zc6Nqq_^;clD_NCcex=@EU$XQ9Pm#NqgeGvd^_aCSl$7zz*Zh%?`E6SUFU#@(2Tm^8
zUUfs;Ou?L&dSYA7%-l33LVAaW?WE>k51uHjS(O#!8@=+q{WAH}&##wV30RdDD&sMa
zb6ycQ&(^c|ulT+6O8ug9{e#BFhCROD7xv%z_gdPb>?Pxmh=#{uQeVnnWu6E;G(VZW
zbm`iHd9n+i1@R;<&b=F`{>1U1jNad!*M*Ka*S`9yKlSVGw`mN5KjtpE%hrG9_m_{E
zTNNsWRLv8ow0Q36di>>=o$sRa%bo}RJ-OkH{Lk%=FD{s$|3&|EJS=s964mC7rhCB2
zBH!E|6eAnVjX=~Bb8`?SYoQOK{47jCRI7y>h&pRw1)?}DT|ksSgqmvU0ODP@^aoKI
zR$#e2D==?AgvV+P=7m{<RnD+B1F5_K;R)G*1w$Z|pM}iiO*WDs`Li})XYtuuf_NZ_
VR9mn_gRKoB$sDl-JNK`xD*y*pH-Z2F

-- 
GitLab


From a364eb51c24fa8dc1c28abfb416244e1eb7cf65f Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Tue, 4 Mar 2025 14:45:23 +0100
Subject: [PATCH 19/35] mutant generation final steps

---
 .../isa.assay.xlsx                            | Bin 9257 -> 9258 bytes
 .../isa.assay.xlsx                            | Bin 11884 -> 11885 bytes
 assays/Metabolomics Analysis/isa.assay.xlsx   | Bin 10745 -> 10746 bytes
 assays/Mutant Generation/isa.assay.xlsx       | Bin 16547 -> 22970 bytes
 .../protocols/R401andEcoliConjugation.md      |   3 +++
 .../SucroseSecondHomologousRecombination.md   |   3 +++
 .../isa.assay.xlsx                            | Bin 11665 -> 11666 bytes
 isa.investigation.xlsx                        | Bin 7875 -> 7876 bytes
 .../isa.study.xlsx                            | Bin 12307 -> 12308 bytes
 .../Plant Growth Conditions/isa.study.xlsx    | Bin 15938 -> 15939 bytes
 10 files changed, 6 insertions(+)
 create mode 100644 assays/Mutant Generation/protocols/R401andEcoliConjugation.md
 create mode 100644 assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md

diff --git a/assays/BGC prediction using antiSMASH/isa.assay.xlsx b/assays/BGC prediction using antiSMASH/isa.assay.xlsx
index b06b8354d92b038d824c16bc5ed5745d5632e109..0c87e3146728ed2f1778c0a9d98276d12929de68 100644
GIT binary patch
delta 724
zcmZ4KvC4xdz?+$ci-CcGgJDn2MxL3BOnYi3FK1K&QkzdR3V^wHndCvD|Cm%kqN>d5
zU{OC7EfBYgWgioWwqjodq9@<uGyqWoTpvK9Ox&};qVsswK-^=zOTpY+0kBb11a`B5
zgagIrgM~jyf`uie_AqkpsR@lb`TR>j{N#Jm+VvTttqNO3mn(&8tq9Fux3|GNDf-)`
z3H#T7R%kf2a{Ju5H$U3VIoqEq_@|*XXtiXgyCA1Sq3)7f=T1~@<&gJyFhTPm*V+WP
ztb&wz_fzjIiHWY*daJ>eQ}2V@sik+$oZ!2-WQx_k=1DszhHLoeZPAsi%GiFzU|)Ll
z0#6a&OwJQ5_0De6hcEE-E!i$ov%!QtdJ9`qu*L$G*1ltlXUyBmv8}|MJL;KJq}LkW
zJ5%PJaI7sU5(o=5U3F~jmGAPG`=6eUw$9QB-K@o@a-PM1Cu`fSXW3iBcLhoAJz;9&
zrK6C4x%P!}`Hx?0eX&&xc47~9OlA8!*Xonj!}A;4cU=mzI3E|c@L3Q~;_BSHf$2{i
z56bBM-Dxg#B)Rt8SN*A9cfU<z5d1NB$z3-8GrxbltlX+lDWp2t!0V7o+|kJ;HS_(J
z&G$VY{x$vZrurxM`_!B3_N}g;`JV|n-Z!VnE&|6XhrB%~@;v2@Kvb=~If&XOuMeUO
zlw~F}D@cNvatdxBDow!(L@iQq0Z}g?l%Apkh*zNK526l2c+5&>AfB}nSPo>;OeL^M
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GBmw}15Gz~&

delta 723
zcmZ4GvC@Mlz?+$ci-CcGgF(7-BhO4mCh5w_%Ndn`)aKKS0$}c4CV7zPKPFX>s4BBM
zSk#Y23&gEr*~bK;t=JcV=*jmu4M3Ct*9VX&6Zb5z=saFE5ce4GQZP4H0BqD0f!%B%
z;Xv{EVBt@aU|~tAJ&c^vm7!4?H^STECf}3RuGbQ2RoE)JTq#s5boITMYEHe*+?(Fc
z|Ms10X3$!GudFQX`0s*kHesnBn6G)R6;qk#qQY==>x5Mi6Skk#nlt%;VsO*csDn)=
z4^9~EKfS|q=e7??TN%5BVjlEP@!lCZL3WX+W@!z(XtD76fO&Vaq7A=X5W8ZqFP-&*
zO4qwg))OrC&Ti3%FZA@KY!Ip0V!|F>#?}<9v5={?@7UxS^R{wqD>CP{dgc`AnZt8u
z%DfYSwIxL&*Gg?yE}whlyZq(;ub;Ecvou0?Yw@X^XYt?3+IH(%e%AVuwMJDFvn!`W
zINe*?e!>0Shp)UczrS$Q=o~22=KW>;^-1WV`N{mgYu6Uc+Z*fjcBzEZ!raTj=}#O_
z%B=glbGy(H=i+x?^{4Xgew)r9_+##pzij?zet-Fxxml@FNOiM;*CCaAM>d<xTE6*a
z`t8r5e{XJ(lKZ*+@yUc4?_b3~+m9UWn^R>MfuodD-X0WpUh+mDs!rY<L~WPX2T_K~
zGLu;pBtcAh1ve0tu3!bC7Av@bsFx5*U(o@?D^&CcQHLNr79}$f&qfI>2Qq1v64<1z
zN?;4WDOrN#4V6tnREDw*QtYo)Hj@XXh0oPB3riUp7<Mo*Fz^G@D*{AJW>QgN`y~ew
F0RXe15W4^X

diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
index e846156dd260e8768b142f4e0c7d8c94e6b7ff3c..13af4a103f05c97a1988a354cd472f29773efa4d 100644
GIT binary patch
delta 768
zcmaD8^EQSjz?+$ci-CcGgJDn2MxN!2OnYi3Z)H>hQk!ow3V^w<ndCvDJj|*fQDbIZ
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diff --git a/assays/Metabolomics Analysis/isa.assay.xlsx b/assays/Metabolomics Analysis/isa.assay.xlsx
index c876c4ee3472462539cb9506cce354ad57473dfa..2828a44d204d086e4ac9ffa61342b8a760a97d20 100644
GIT binary patch
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diff --git a/assays/Mutant Generation/isa.assay.xlsx b/assays/Mutant Generation/isa.assay.xlsx
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diff --git a/assays/Mutant Generation/protocols/R401andEcoliConjugation.md b/assays/Mutant Generation/protocols/R401andEcoliConjugation.md
new file mode 100644
index 0000000..75c4ee8
--- /dev/null
+++ b/assays/Mutant Generation/protocols/R401andEcoliConjugation.md	
@@ -0,0 +1,3 @@
+## Conjugation of E. coli and R401 and selection for first homologous recombination event
+
+*E. coli* BW29427 cells containing the plasmid and R401 were inoculated into 4 ml of 50% TSB containing 25 μg/ml Kan and 50 μg/ml DAP or 50% TSB and incubated overnight at 37 °C with 180 rpm agitation or 25 °C with 180 rpm agitation, respectively. Cells were harvested by centrifugation at 8,000 rpm for 2 min at room temperature, then washed 3x and subsequently resuspended in 1 ml of 50% TSB followed by centrifugation, after which the supernatant was discarded. After quantifying OD600, both cultures were mixed to equal parts and approx. 10x concentrated by centrifugation. The bacterial suspension was plated on 50% TSA plates containing 50 μg/ml DAP and incubated at 25 °C overnight to allow for conjugation events. The mating patches were scraped of the plate and resuspended in 1 ml 50% TSB. Then, 100 μl were spread on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Nitrofurantoin (Nitro; Sigma-Aldrich; to counter-select *E. coli*) and incubated at 25 °C. Colonies were validated for successful genomic insertion of the plasmid via colony PCR using a primer specific to the genomic DNA approx. 150 bp upstream of the upward flanking region (upup) and the plasmid specific M13F primer. Colony PCR was performed on at least 15 separate colonies and a WT control with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously, but with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis followed by Sanger sequencing following the manufacturer's protocol.
\ No newline at end of file
diff --git a/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md
new file mode 100644
index 0000000..dcfd4b1
--- /dev/null
+++ b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md	
@@ -0,0 +1,3 @@
+## Sucrose counter-selection to induce the second homologous recombination event
+
+A R401 colony with a successful genomic insertion of the plasmid was resuspended from a plate into 1 ml of 50% TSB. The cell density in the medium was then measured using the Multisizer 4e Coulter Counter (Beckman Coulter) following the manufacturer's protocol. One hundred microliters of 500 cells/μl, 5,000 cells/μl and 50,000 cells/μl dilutions were spread on three separate 50% TSA plates containing 300 mM sucrose. The plates were incubated at 25 °C for approx. 48 h. At least 30 colonies were examined by colony PCR using the respective upup and dwdw primers. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol. They were also streaked on 50% TSA containing 25 μg/ml Kan to verify loss of the plasmid. A second colony PCR was performed on positive colonies and a wt control to validate the absence of the GOI, using a forward (inF) and reverse (inR) primer inside the GOI. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25 μl reactions as described previously. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification, 4 ml of 50% TSB were inoculated with a positive colony and grown overnight at 25 °C at 180 rpm. Finally, 750 μl of the overnight culture were added to 750 μl of 50% glycerol in an internally threaded 1.8 ml Nunc CryoTube, gently mixed, and stored at -80 °C.
\ No newline at end of file
diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx
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GIT binary patch
delta 761
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delta 759
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
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delta 629
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delta 628
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diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx
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GIT binary patch
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delta 771
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-- 
GitLab


From cbda55bc8ef8787c913e9051355ae1eff6fbf808 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Tue, 4 Mar 2025 16:07:32 +0100
Subject: [PATCH 20/35] added protocol and assay for MiniTn5forR401andR569

---
 .gitattributes                                  |   1 +
 .../README.md                                   |   0
 .../dataset/.gitkeep                            |   0
 .../isa.assay.xlsx                              | Bin 0 -> 10631 bytes
 .../protocols/.gitkeep                          |   0
 .../Mini-Tn5TransposonMutantInR401andR569.md    |   3 +++
 6 files changed, 4 insertions(+)
 create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/README.md
 create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep
 create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/isa.assay.xlsx
 create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep
 create mode 100644 assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md

diff --git a/.gitattributes b/.gitattributes
index 6608dce..7ca77f5 100644
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+++ b/.gitattributes
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 assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
+assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/README.md b/assays/Mini-Tn5TransposonMutantInR401andR569/README.md
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diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md
new file mode 100644
index 0000000..b4a2866
--- /dev/null
+++ b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md
@@ -0,0 +1,3 @@
+## Establishment of mini-Tn5 transposon mutant collections in R401 and R569
+
+Mini Tn5-mutant collections of R401 and R569 were established similarly with only minor changes as described below. Liquid cultures of R401 or R569 and *E. coli* strain BW29427 carrying plasmid pUTmTn5Km2 (15) were grown overnight in no antibiotics or 25 μg/ml Kan and 50 μg/ml DAP at 25 °C or 37 °C, respectively. Conjugation was carried out as described above in *“Conjugation of E. coli and R401 and selection for first homologous recombination event”*. For R401, the mating patch was taken up in 1 ml 50% TSB liquid medium and subsequently plated on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Zeocin in four different dilutions (undiluted, 1:3, 1:4, and 1:5) and left to grow at 25 °C for 48 h. Individual colonies were picked in 100 μl sterile 50% TSB in 96-well culture plates, sealed and left to grow at 25 °C and 180 rpm for 24 h. Subsequently, 100 μl 50% glycerol were added to each well and plates were frozen until further processing. The outer rows and columns were left uninoculated as to avoid positional effects in the subsequent forward genetic screen. For R569, resuspended mating patches were stocked at -80 °C in 700-μl aliquots using a final concentration of 25% Glycerol. 1:4 dilutions were plated onto 50% TSA plates supplemented with 120 μg/ml Kan, 50 μg/ml Rifampicin and 50 μg/ml Zeocin and incubated at 25 °C for 48h. Individual colonies were inoculated in 100 μl 50% TSB supplemented with the same antibiotics at the same concentrations in 96-well plates and incubated at 25 °C and 180 rpm for 48 h. Then, 100 μl 50% glycerol were added to each culture and plates were frozen at -80 °C.
\ No newline at end of file
-- 
GitLab


From fad359aedaa9c851e05c2d3b6b623ab23263c3e7 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Tue, 4 Mar 2025 17:04:14 +0100
Subject: [PATCH 21/35] added protocol and dataset for loss of R401s growth
 inhibition of Rs GMI1600

---
 .gitattributes                                   |   1 +
 .../README.md                                    |   0
 .../dataset/.gitkeep                             |   0
 .../dataset/Table S5.xlsx                        |   3 +++
 .../isa.assay.xlsx                               | Bin 0 -> 6926 bytes
 .../protocols/.gitkeep                           |   0
 ...n5ScreenLossR401sGrowthInhibitionRsGMI1600.md |   5 +++++
 7 files changed, 9 insertions(+)
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/.gitkeep
 create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md

diff --git a/.gitattributes b/.gitattributes
index 7ca77f5..51fdd51 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -5,3 +5,4 @@ _publication/dataverse_files/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=l
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 assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx
new file mode 100644
index 0000000..9d758ba
--- /dev/null
+++ b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx	
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diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/.gitkeep b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md
new file mode 100644
index 0000000..a1d29b1
--- /dev/null
+++ b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md
@@ -0,0 +1,5 @@
+## Mini-Tn5 transposon mutant screen for loss of R401s growth inhibition of Rs GMI1600
+
+Each R401 mini-Tn5 transposon mutant was screened individually for loss of inhibitory activity against GFP-expressing *Rs* GMI1600 and for wild type-like growth, as we observed in first trials that mutants that are impaired in growth are also more likely to have reduced inhibitory activity against Rs GMI1600. This screen was conducted in a 96-well plate format with GFP expression of *Rs* GMI1600 in response to individual R401 mutants  and Abs~600~ of axenically grown, individual R401 mutants as readouts. Therefore, for each mutant, two wells were inoculated in parallel, one in the presence of *Rs* GMI1600 and one grown axenically. Rs GMI1600 was streaked on 50% TSA plates and left to grow at 25 °C for 96 h. The same day, R401 mutants were each inoculated into 150 μl Artificial Root Exudates (ARE; **Table S5**) in 96-well culture plates (‘R401 preculture plates’) from glycerol stocks using a multi-stamp replicator and left to grow at 25 °C and 150 rpm for 96 h until saturation. Then, a *Rs* GMI1600 preculture was inoculated into 10 ml ARE and grown overnight at 25 °C and 150 rpm. Approximately 24 h later, the preculture was 1:10 diluted with 90 ml ARE and left to grow under the same conditions. Approximately 24 h later, the 100 ml *Rs* GMI1600 culture was concentrated by centrifugation at 4,000 rpm for 15 min, washed 3x and resuspended in ARE. Subsequently, OD600 was quantified and set to 0.2 in ARE. Then, 75 μl of this *Rs* GMI1600 suspension were transferred to each well of a sterile 96-well bacterial culture plate (Greiner-CELLSTAR-96-Well plate, transparent, flatbottom; Sigma-Aldrich) per ‘R401 preculture plate’, referred to as the ‘R401-*Rs* interaction plate’. Per ‘R401 preculture plate’ one sterile 96-well bacterial culture plate (Greiner-CELLSTAR-96-Well plate, transparent, flatbottom; Sigma-Aldrich) was filled with 150 μl ARE per well and R401 mutants were inoculated from the ‘R401 preculture plate’ using a multi-stamp replicator; this plate is referred to as the ‘axenic R401 plate’. After mixing by pipetting, 75 μl R401 mutant suspension were transferred from the ‘axenic R401 plate’ to the ‘R401-Rs interaction plate’. Finally, 75 μl ARE were added to each well of the ‘axenic R401 plate’. The ‘axenic R401 plate’ and ‘R401-Rs interaction plate’ thereby contain the same volume and concentration of R401 mutants, while the latter also contains a final concentration of OD~600~ 0.1 *Rs* GMI1600 per well. Both plates were closed with a lid and incubated at 25 °C and 150 rpm for 48 h. Subsequently, Abs~600~ was measured for the ‘axenic R401 plate’ and GFP fluorescence was quantified at 𝜆~excitation~=475 nm and 𝜆~emission~=510 nm for the ‘R401-Rs interaction plate’. Both measurements were taken using a microplate reader (Infinite M200 PRO, Tecan). Subsequently, candidate R401 mutants were selected based on two criteria: (I) loss of *Rs* GMI1600 inhibition; a mutant was considered a candidate if the GFP fluorescence in the ‘R401-Rs interaction plate’ was lower than 3-fold the absolute deviation around the median (MAD) (16) compared to the respective plates median, and (II) wild-type-like growth; a mutant was considered a candidate if the Abs~600~ in the ‘axenic R401 plate’ was not lower than 3x MAD compared to the respective plate’s median. Candidate R401 mutants were freshly picked from glycerol stocks and validated twice more independently using the same assay. Finally, 38 mutants that showed wild-type-like growth and robustly reduced inhibitory activity against *Rs* GMI1600 were subsequently tested in an orthogonal mBA experiment with Rs as target bacterium, as described before.
+
+(16) C. Leys, C. Ley, O. Klein, P. Bernard, L. Licata, Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median. *J Exp Soc Psychol* **49**, 764–766 (2013).
\ No newline at end of file
-- 
GitLab


From 815727b01007c16aafb9f5fdf3c79b0b10126c35 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 13 Mar 2025 12:13:25 +0100
Subject: [PATCH 22/35] minitrn loss of R401

---
 .../isa.assay.xlsx                            | Bin 6926 -> 15558 bytes
 1 file changed, 0 insertions(+), 0 deletions(-)

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-- 
GitLab


From f8a9400cb36ec0944ab5064d8c332e6d5955c35e Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 13 Mar 2025 14:41:29 +0100
Subject: [PATCH 23/35] Mini-Tn5ScreenPyoverdineLackR569 assay and protocol

---
 .gitattributes                                |   1 +
 .../Mini-Tn5ForPyoverdineLackofR569/README.md |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 10297 bytes
 .../protocols/.gitkeep                        |   0
 ...Tn5TransposonScreenPyoverdineLackofR569.md |   3 +++
 .../isa.assay.xlsx                            | Bin 15558 -> 15561 bytes
 7 files changed, 4 insertions(+)
 create mode 100644 assays/Mini-Tn5ForPyoverdineLackofR569/README.md
 create mode 100644 assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep
 create mode 100644 assays/Mini-Tn5ForPyoverdineLackofR569/isa.assay.xlsx
 create mode 100644 assays/Mini-Tn5ForPyoverdineLackofR569/protocols/.gitkeep
 create mode 100644 assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md

diff --git a/.gitattributes b/.gitattributes
index 51fdd51..ed16a26 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -6,3 +6,4 @@ assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:sp
 assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/README.md b/assays/Mini-Tn5ForPyoverdineLackofR569/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep b/assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/isa.assay.xlsx b/assays/Mini-Tn5ForPyoverdineLackofR569/isa.assay.xlsx
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diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/.gitkeep b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md
new file mode 100644
index 0000000..9aba88c
--- /dev/null
+++ b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md
@@ -0,0 +1,3 @@
+## Mini-Tn5 transposon mutant screen for lack of pyoverdine fluorescence of R569
+
+R569 mini-Tn5 mutants were cultured in 96 well plates at 25 °C and 180 rpm for five days in 50% TSB medium. For the initial mutant screen, fluorescence was acquired at 𝜆~excitation~=395 nm and 𝜆~emission~=470 nm in a microplate reader (Infinite M200 PRO, Tecan). Out of ~2,000 mutants analysed, the fluorescence-based screening identified twelve R569 mutants that showed severely reduced pyoverdine-specific fluorescence but retained median-like growth behaviour (Thresholds: -6x MAD fluorescence, >-1x MAD Abs~600~). For validation, cultures were pre-grown for five days in 50% TSB before sub-culturing into fresh siderophore medium (see **Table S5**) and growth for five additional days. Finally, bacterial culture density (Abs~600~) was determined, and pyoverdine-specific fluorescence of bacterial culture supernatants was captured at 𝜆~excitation~=410 nm and 𝜆~emission~=500 nm. For  comparison between genotypes, fluorescence capacity was calculated by dividing the pyoverdine-specific fluorescence by culture density.
\ No newline at end of file
diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx
index 5a8d5b32ec6e5e437c862b391b832e4d97436c3b..917c8436bc6717c1ecbf1b59232d204bad7bfb32 100644
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-- 
GitLab


From 9c7f90376c32d09e1dc0c7e2b3fcc25cca15db95 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 13 Mar 2025 15:26:43 +0100
Subject: [PATCH 24/35] R401and R569 integration pcr and protocol

---
 .gitattributes                                  |   1 +
 .../README.md                                   |   0
 .../dataset/.gitkeep                            |   0
 .../isa.assay.xlsx                              | Bin 0 -> 10248 bytes
 .../protocols/.gitkeep                          |   0
 ...entificationofIntegrationSitesR401andR569.md |   6 ++++++
 6 files changed, 7 insertions(+)
 create mode 100644 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/README.md
 create mode 100644 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep
 create mode 100644 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/isa.assay.xlsx
 create mode 100644 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/.gitkeep
 create mode 100644 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/Mini-Tn5IdentificationofIntegrationSitesR401andR569.md

diff --git a/.gitattributes b/.gitattributes
index ed16a26..d99cf23 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -7,3 +7,4 @@ assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Tabl
 assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/README.md b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/isa.assay.xlsx b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/isa.assay.xlsx
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diff --git a/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/.gitkeep b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/Mini-Tn5IdentificationofIntegrationSitesR401andR569.md b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/Mini-Tn5IdentificationofIntegrationSitesR401andR569.md
new file mode 100644
index 0000000..397e325
--- /dev/null
+++ b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/Mini-Tn5IdentificationofIntegrationSitesR401andR569.md
@@ -0,0 +1,6 @@
+## Identification of mini-Tn5 transposon integration sites in the genomes of R401 and R569
+
+The chromosomal mini-Tn5 transposon integration sites in the R401 or R569 genomes were determined similarly as described before (15). Briefly, a colony from each strain was resuspended in 20 μl of sterile deionized water. Subsequently, a two-step PCR (TAIL-PCR) was performed starting with an arbitrarily primed PCR (PCR1), followed by a nested PCR (PCR2) on the generated product using the primers as described (**Table S4**). PCR reactions were conducted using the BIORON DFS-Taq polymerase reaction kit according to the manufacturer’s instructions. One microliter of a resuspended bacterial colony was used as template for PCR1 in a total PCR reaction volume of 25 μl. PCR1 was conducted using primers JO4 and JO28 and the following steps were applied: 95 °C for 5 min, six
+cycles of 95 °C for 15 sec, 30 °C for 30 sec and 1 min elongation at 72 °C, followed by 30 cycles with 95 °C for 15 seconds, 45 °C for 30 seconds and 1 min elongation at 72 °C. Final elongation was for 5 min at 72°C. PCR2 was conducted using 0.3 μl of the PCR1 product and the primers JO1 and JO5. The following steps were applied: 95 °C for 5 min followed by 30 cycles of 95 °C for 15 sec, 57 °C for 30 sec and 1 min elongation at 72 °C.
+Final elongation was for 5 min at 72°C. R401 or R569 wild type was included to account for unspecific amplifications. PCR products were separated by agarose gel electrophoresis. One of the most prominent bands from each sample was extracted using the Nucleospin Gel and PCR clean-up kit (Macherey-Nagel). The PCR product was eluted into a final buffer volume of 20 μl; DNA concentration was determined with a spectrophotometer (NanoDrop One, Thermo Scientific), and 75 ng of each product were sent for Sanger 
+ sequencing (Eurofins genomics). Finally, chromosomal Tn5 integration sites were assessed by alignment of the obtained sequences flanked by the integrated Tn5 transposon in the R401 or R569 genome using the Geneious Prime or CLC Genomics Workbench software, respectively (Qiagen Digital Insights). Matching open reading frames (ORFs) were further aligned to the NCBI BLAST (17) nucleotide and protein data base (using BLASTn and BLASTp algorithm).
\ No newline at end of file
-- 
GitLab


From 95ca08ee29431d6f8a30fbaeec3ef633cd2a6ebf Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 13 Mar 2025 15:47:59 +0100
Subject: [PATCH 25/35] R401pvdY Complementation assay and protocol

---
 .gitattributes                                   |   1 +
 assays/ComplementationR401pvdY/README.md         |   0
 assays/ComplementationR401pvdY/dataset/.gitkeep  |   0
 assays/ComplementationR401pvdY/isa.assay.xlsx    | Bin 0 -> 9425 bytes
 .../ComplementationR401pvdY/protocols/.gitkeep   |   0
 .../ComplementationR401\342\210\206pvdy.md"      |   5 +++++
 6 files changed, 6 insertions(+)
 create mode 100644 assays/ComplementationR401pvdY/README.md
 create mode 100644 assays/ComplementationR401pvdY/dataset/.gitkeep
 create mode 100644 assays/ComplementationR401pvdY/isa.assay.xlsx
 create mode 100644 assays/ComplementationR401pvdY/protocols/.gitkeep
 create mode 100644 "assays/ComplementationR401pvdY/protocols/ComplementationR401\342\210\206pvdy.md"

diff --git a/.gitattributes b/.gitattributes
index d99cf23..fb6ffed 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -8,3 +8,4 @@ assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lf
 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/ComplementationR401pvdY/README.md b/assays/ComplementationR401pvdY/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/ComplementationR401pvdY/dataset/.gitkeep b/assays/ComplementationR401pvdY/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/ComplementationR401pvdY/isa.assay.xlsx b/assays/ComplementationR401pvdY/isa.assay.xlsx
new file mode 100644
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diff --git a/assays/ComplementationR401pvdY/protocols/.gitkeep b/assays/ComplementationR401pvdY/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git "a/assays/ComplementationR401pvdY/protocols/ComplementationR401\342\210\206pvdy.md" "b/assays/ComplementationR401pvdY/protocols/ComplementationR401\342\210\206pvdy.md"
new file mode 100644
index 0000000..8432656
--- /dev/null
+++ "b/assays/ComplementationR401pvdY/protocols/ComplementationR401\342\210\206pvdy.md"
@@ -0,0 +1,5 @@
+## Complementation of R401 ∆pvdy
+
+Complementation of R401 *∆pvdy* was conducted by expressing the coding region of R401 *pvdY* under its putative native promoter from a low-copy plasmid in the *∆pvdy* mutant background. Plasmid construction was conducted using Gibson assembly as described before. In brief, the coding region of R401 *pvdY* was co amplified with a 1.5 kb region upstream of the gene and ligated into a linearized pSEVA22l (18) vector backbone. This vector was integrated into *E. coli* BW29427 cells and subsequently integrated into R401 *∆pvdy via* bi-parental conjugation.
+
+(18) E. Martínez-García, *et al.*, SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts. *Nucleic Acids Res* **48**, D1164–D1170 (2020).
\ No newline at end of file
-- 
GitLab


From 97f288cc0d6bfafb0bf617a9bcd1ee845945aa18 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 13 Mar 2025 16:07:15 +0100
Subject: [PATCH 26/35] iron mobilization protocol

---
 .gitattributes                                   |   1 +
 assays/InvitroIronMobilizationAssay/README.md    |   0
 .../dataset/.gitkeep                             |   0
 .../InvitroIronMobilizationAssay/isa.assay.xlsx  | Bin 0 -> 6907 bytes
 .../protocols/.gitkeep                           |   0
 .../protocols/InvitroIronMobilizationAssay.md    |  11 +++++++++++
 6 files changed, 12 insertions(+)
 create mode 100644 assays/InvitroIronMobilizationAssay/README.md
 create mode 100644 assays/InvitroIronMobilizationAssay/dataset/.gitkeep
 create mode 100644 assays/InvitroIronMobilizationAssay/isa.assay.xlsx
 create mode 100644 assays/InvitroIronMobilizationAssay/protocols/.gitkeep
 create mode 100644 assays/InvitroIronMobilizationAssay/protocols/InvitroIronMobilizationAssay.md

diff --git a/.gitattributes b/.gitattributes
index fb6ffed..9c182c3 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -9,3 +9,4 @@ assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filte
 assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/InvitroIronMobilizationAssay/README.md b/assays/InvitroIronMobilizationAssay/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/InvitroIronMobilizationAssay/dataset/.gitkeep b/assays/InvitroIronMobilizationAssay/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/InvitroIronMobilizationAssay/isa.assay.xlsx b/assays/InvitroIronMobilizationAssay/isa.assay.xlsx
new file mode 100644
index 0000000000000000000000000000000000000000..914bb893093a07c87af3c9eb215778a134aa91bb
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HcmV?d00001

diff --git a/assays/InvitroIronMobilizationAssay/protocols/.gitkeep b/assays/InvitroIronMobilizationAssay/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/InvitroIronMobilizationAssay/protocols/InvitroIronMobilizationAssay.md b/assays/InvitroIronMobilizationAssay/protocols/InvitroIronMobilizationAssay.md
new file mode 100644
index 0000000..b9dd7cf
--- /dev/null
+++ b/assays/InvitroIronMobilizationAssay/protocols/InvitroIronMobilizationAssay.md
@@ -0,0 +1,11 @@
+## In vitro iron mobilization assay
+
+The capability of R401 and R569 mutants of solubilizing inaccessible ferric iron was tested using a previously described photometric assay (19). Bacteria were cultured at 25 °C and 180 rpm for five days in 50% TSB and subsequently subcultured and 1:50 diluted in siderophore medium (**Table S5**). Diluted cultures were grown for an additional five days under the same conditions. At five days post inoculation (dpi), bacterial load was quantified by OD600 determination. Subsequently, cells were pelleted by centrifugation for 15 min at 4,000 rpm. The cell-free supernatant was diluted 5-fold with 10 mM MgCl2 and 25 μl were mixed with 100 μl of CAS assay solution (20) in three technical replicates and incubated for approx. 40 min at room temperature in the dark. Using an Infinite 200 PRO plate reader (TECAN), Abs636 was measured as an indication for the transition from complexed iron (blue complex) to solubilized or siderophore-bound iron (yellow). Finally, bacterial iron mobilizing capacity was computed according to the following formula:
+
+
+Fe-mobilizing capacity [μM Fe / OD₆₀₀] =  
+((Abs₆₃₆(Medium) − Abs₆₃₆(Sample)) / 
+Abs₆₃₆(Sample)) × 2 nmol  ───────────────────────────── 
+(Volume(Supernatant) × OD₆₀₀)
+
+
-- 
GitLab


From b2243d57928debda0c3c90acf1910af520430d47 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 14 Mar 2025 12:05:47 +0100
Subject: [PATCH 27/35] Iron Mobilization assay

---
 .../isa.assay.xlsx                            | Bin 6907 -> 10874 bytes
 1 file changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/InvitroIronMobilizationAssay/isa.assay.xlsx b/assays/InvitroIronMobilizationAssay/isa.assay.xlsx
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-- 
GitLab


From 4c800b98e68ad081ae70441dcdea788d9e6190e4 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 14 Mar 2025 12:35:53 +0100
Subject: [PATCH 28/35] Bacterial Grwoth rate assay and protocol, some changes
 in tables column names

---
 .gitattributes                                |   1 +
 assays/BacterialGrowthRates/README.md         |   0
 assays/BacterialGrowthRates/dataset/.gitkeep  |   0
 assays/BacterialGrowthRates/isa.assay.xlsx    | Bin 0 -> 10078 bytes
 .../BacterialGrowthRates/protocols/.gitkeep   |   0
 .../BacterialGrowthRatesValidation.md         |   6 ++++++
 assays/ComplementationR401pvdY/isa.assay.xlsx | Bin 9425 -> 9445 bytes
 .../isa.assay.xlsx                            | Bin 10874 -> 10874 bytes
 .../isa.assay.xlsx                            | Bin 10297 -> 10298 bytes
 .../isa.assay.xlsx                            | Bin 15561 -> 15566 bytes
 10 files changed, 7 insertions(+)
 create mode 100644 assays/BacterialGrowthRates/README.md
 create mode 100644 assays/BacterialGrowthRates/dataset/.gitkeep
 create mode 100644 assays/BacterialGrowthRates/isa.assay.xlsx
 create mode 100644 assays/BacterialGrowthRates/protocols/.gitkeep
 create mode 100644 assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md

diff --git a/.gitattributes b/.gitattributes
index 9c182c3..a008537 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -10,3 +10,4 @@ assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merg
 assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/BacterialGrowthRates/README.md b/assays/BacterialGrowthRates/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/BacterialGrowthRates/dataset/.gitkeep b/assays/BacterialGrowthRates/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/BacterialGrowthRates/isa.assay.xlsx b/assays/BacterialGrowthRates/isa.assay.xlsx
new file mode 100644
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diff --git a/assays/BacterialGrowthRates/protocols/.gitkeep b/assays/BacterialGrowthRates/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md b/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md
new file mode 100644
index 0000000..74bb5be
--- /dev/null
+++ b/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md
@@ -0,0 +1,6 @@
+## Validation of bacterial growth rates
+
+The growth of individual R401 mutants and wild type was assessed by continuously measuring OD~600~ of actively growing bacterial cultures in an Infinite 200 PRO plate reader (TECAN) over 48 h at 25 °C and approx. 300 rpm. Overnight cultures in 50% TSB were pelleted and washed as described before. Subsequently, OD~600~ was measured and set to 0.02 in either artificial root exudates (ARE) or ARE lacking FeCl~3~. Composition of ARE
+was adopted from (21) and can be found in **Table S5**.
+
+(21) E. Baudoin, E. Benizri, A. Guckert, Impact of artificial root exudates on the bacterial community structure in bulk soil and maize rhizosphere. *Soil Biol Biochem* **35**, 1183–1192 (2003).
\ No newline at end of file
diff --git a/assays/ComplementationR401pvdY/isa.assay.xlsx b/assays/ComplementationR401pvdY/isa.assay.xlsx
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delta 2357
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diff --git a/assays/InvitroIronMobilizationAssay/isa.assay.xlsx b/assays/InvitroIronMobilizationAssay/isa.assay.xlsx
index a624fe563a8f60b198c14347210335914fda4cc5..8b7c48f3c475ca9e2b3b371eff4c63ac50935883 100644
GIT binary patch
delta 957
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delta 957
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diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/isa.assay.xlsx b/assays/Mini-Tn5ForPyoverdineLackofR569/isa.assay.xlsx
index 7d12f4eeed689a3daf17236b1ca1b31321848bc0..c2f14e9080aa436204cab2e587274e8cd1e11795 100644
GIT binary patch
delta 1842
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delta 1841
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diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx
index 917c8436bc6717c1ecbf1b59232d204bad7bfb32..2b7311be7d0b7b6d8908400855da5600c0522517 100644
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-- 
GitLab


From 045e3ba5a267d518110dfd50557673163ccc87a1 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 14 Mar 2025 14:58:27 +0100
Subject: [PATCH 29/35] Microbiota reconstruction flowpot assay

---
 .gitattributes                                  |   1 +
 .../MicrobiotaReconstitutionFlowpot/README.md   |   0
 .../dataset/.gitkeep                            |   0
 .../isa.assay.xlsx                              | Bin 0 -> 12814 bytes
 .../protocols/.gitkeep                          |   0
 .../MicrobiotaReconstitutionFlowpot.md          |   5 +++++
 6 files changed, 6 insertions(+)
 create mode 100644 assays/MicrobiotaReconstitutionFlowpot/README.md
 create mode 100644 assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep
 create mode 100644 assays/MicrobiotaReconstitutionFlowpot/isa.assay.xlsx
 create mode 100644 assays/MicrobiotaReconstitutionFlowpot/protocols/.gitkeep
 create mode 100644 assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md

diff --git a/.gitattributes b/.gitattributes
index a008537..50a354a 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -11,3 +11,4 @@ assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filt
 assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/MicrobiotaReconstitutionFlowpot/README.md b/assays/MicrobiotaReconstitutionFlowpot/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep b/assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/MicrobiotaReconstitutionFlowpot/isa.assay.xlsx b/assays/MicrobiotaReconstitutionFlowpot/isa.assay.xlsx
new file mode 100644
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diff --git a/assays/MicrobiotaReconstitutionFlowpot/protocols/.gitkeep b/assays/MicrobiotaReconstitutionFlowpot/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md b/assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md
new file mode 100644
index 0000000..cdf67a0
--- /dev/null
+++ b/assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md
@@ -0,0 +1,5 @@
+## Microbiota reconstitution in the gnotobiotic Flowpot system
+
+Flowpot assembly was performed according to (24) with minor adjustments. A 2:1 mixture of peat potting mix and vermiculite was used as a matrix. The matrix was sterilized two times (25 min liquid cycle (121 °C) and 45 min solid cycle (134 °C)) and stored at 60 °C until completely dry. Prior to Flowpot assembly, the matrix was rehydrated with sterile MiliQ water. Flowpots were assembled by adding a layer of glass beads to the conical end of a truncated syringe, followed by a layer of the rehydrated, sterile substrate, subsequently covered with a sterile mesh secured by a cable tie. Assembled Flowpots were sterilized on a 25 min liquid cycle, stored at 60 °C overnight and sterilized twice on a 45 min solid cycle. Bacterial strains, cultivated as described before, were harvested, 3x washed and pooled in equal ratios. Then, 1.25 ml bacterial pool (OD~600~ 1.0) were added to 500 ml of 1⁄2 MS medium for a final bacterial OD~600~ of 0.0025. Flowpots were first flushed with sterile MiliQ water, then inoculated with 50 ml of 1⁄2 MS. Eight inoculated Flowpots were placed into each sterile microbox (TP1200, Sac O2) and stored at room temperature overnight. Exactly five sterilized seeds were sown per Flowpot and left to grow under the previously described  conditions. At 21 dpi, shoot fresh weight was measured individually for each plant. Roots from a single Flowpot were thoroughly cleaned from soil particles in sterile water using tweezers. Six representative Flowpots were selected for harvesting root and matrix samples. Cleaned roots from each Flowpot were pooled in 2 ml lysing matrix E tubes (MP Biomedicals), snap-frozen and stored at -80 °C until further use. Additionally, <100 mg of soil were taken from each Flowpot, snap-frozen and stored in weighed 2 ml lysing matrix E tube (MP Biomedicals) at -80 °C until further use.
+
+(24) J. M. Kremer, et al., Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research. *Nature Protocols 2021 16:5* **16**, 2450–2470 (2021).
\ No newline at end of file
-- 
GitLab


From 094fd9794ff62bda03e9c44e3410d6f5e0adf015 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 14 Mar 2025 16:06:05 +0100
Subject: [PATCH 30/35] Athaliana seedlings

---
 .gitattributes                                |   1 +
 assays/BacterialGrowthRates/isa.assay.xlsx    | Bin 10078 -> 10079 bytes
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 14425 bytes
 .../protocols/.gitkeep                        |   0
 .../R401onAthalianaSeedlingsAgarPlates.md     |   3 +++
 7 files changed, 4 insertions(+)
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/dataset/.gitkeep
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md

diff --git a/.gitattributes b/.gitattributes
index 50a354a..e09f562 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -12,3 +12,4 @@ assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -t
 assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Mono-association[[:space:]]experiment[[:space:]]of[[:space:]]R401[[:space:]]on[[:space:]]Athaliana[[:space:]]seedlings[[:space:]]in[[:space:]]agar[[:space:]]plates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
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diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
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index 0000000..e69de29
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/dataset/.gitkeep b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx
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diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md
new file mode 100644
index 0000000..c7878ba
--- /dev/null
+++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md	
@@ -0,0 +1,3 @@
+## Mono-association experiment of R401 on A. thaliana seedlings in agar plates
+
+This protocol is adapted from (25). In brief, *A. thaliana* seeds were sterilized, germinated, and transferred to 1⁄2 MS agar plates without sucrose, as described before. After transfer of seedlings, plants were grown for another 14 days under the same conditions. R401 wild-type and mutants were grown in 50% TSB overnight as described before. Bacterial cells were pelleted by centrifugation, washed 3x in 10 mM MgCl2 and OD~600~ was measured and adjusted to 0.0001. Agar plates were flushed with 15 ml of bacterial suspension for 5 min. The bacterial suspension were removed, and plants were carefully transferred to new 1⁄2 MS agar plates. After 24 h, roots were cut using a sterile scalpel and collected in pre-weighed, sterile 2 ml tubes containing 1 steel bead (3 mm diameter). Tubes were weighed again to assess the root fresh weight. Subsequently, roots were ground in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm at 15 s intervals. Then, 250 μl of sterile 10 mM MgCl2 were added to each tube and roots were ground again under the same conditions. Each sample was subsequently 5x 1:10 diluted in sterile 10 mM MgCl2. Undiluted samples and each dilution were plated on 50% TSA square plates, dried and left to grow at 25 °C until single colonies appeared. Pictures were taken and single colonies were counted blinded.
\ No newline at end of file
-- 
GitLab


From 0caf288ce2a645580b5ea038d85e95197c61f725 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 14:21:25 +0100
Subject: [PATCH 31/35] assays for monoassociation seedlings and microbiota
 reconstitution

---
 .../isa.assay.xlsx                            | Bin 12814 -> 9732 bytes
 .../isa.assay.xlsx                            | Bin 14425 -> 13480 bytes
 2 files changed, 0 insertions(+), 0 deletions(-)

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diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx
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-- 
GitLab


From c8f268788fdbc3ed16437095beb365dcff3ce95a Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 14:53:08 +0100
Subject: [PATCH 32/35] DNA Isolation

---
 .gitattributes                                |   1 +
 assays/DNA Isolation/README.md                |   0
 assays/DNA Isolation/dataset/.gitkeep         |   0
 assays/DNA Isolation/isa.assay.xlsx           | Bin 0 -> 9419 bytes
 assays/DNA Isolation/protocols/.gitkeep       |   0
 .../DNA Isolation/protocols/DNAIsolation.md   |   4 ++++
 .../isa.assay.xlsx                            | Bin 13480 -> 10656 bytes
 7 files changed, 5 insertions(+)
 create mode 100644 assays/DNA Isolation/README.md
 create mode 100644 assays/DNA Isolation/dataset/.gitkeep
 create mode 100644 assays/DNA Isolation/isa.assay.xlsx
 create mode 100644 assays/DNA Isolation/protocols/.gitkeep
 create mode 100644 assays/DNA Isolation/protocols/DNAIsolation.md

diff --git a/.gitattributes b/.gitattributes
index e09f562..eac0854 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -13,3 +13,4 @@ assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=l
 assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mono-association[[:space:]]experiment[[:space:]]of[[:space:]]R401[[:space:]]on[[:space:]]Athaliana[[:space:]]seedlings[[:space:]]in[[:space:]]agar[[:space:]]plates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/DNA Isolation/README.md b/assays/DNA Isolation/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/DNA Isolation/dataset/.gitkeep b/assays/DNA Isolation/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/DNA Isolation/isa.assay.xlsx b/assays/DNA Isolation/isa.assay.xlsx
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diff --git a/assays/DNA Isolation/protocols/.gitkeep b/assays/DNA Isolation/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/DNA Isolation/protocols/DNAIsolation.md b/assays/DNA Isolation/protocols/DNAIsolation.md
new file mode 100644
index 0000000..abb6f07
--- /dev/null
+++ b/assays/DNA Isolation/protocols/DNAIsolation.md	
@@ -0,0 +1,4 @@
+## DNA isolation
+
+For DNA extractions, root and soil samples were homogenized in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm with 15 s intervals. DNA was extracted with a modified, high-throughput version of the FastDNA SPIN kit for Soil (MP Biomedicals). In brief, samples were taken up in sodium phosphate buffer (MP Biomedicals) and MT buffer (MP Biomedicals), then homogenized as described before.
+After centrifugation for 15 min at 13,000 rpm, 150 μl of the supernatant were transferred to a 96-well Acroprep Advance filter plate (with 0.2 μm Supor filter; Pall). Once full, the filter plate was positioned on a PCR plate filled with 50 μl Binding Matrix (MP Biomedicals) per well and centrifuged for 15 min to remove residual soil particles. This and all subsequent centrifugation steps were carried out in a swing out centrifuge at 1,500 rpm. The PCR plate was sealed and shaken for 3 min to allow binding of the DNA to the Binding Matrix. The suspension was pipetted onto a second filter plate of the same kind, positioned on a collection plate, and centrifuged for 15 min. The flowthrough was discarded. Then, 200 μl SEWS-M washing buffer (MP Biomedicals) were pipetted into each well of the filter plate and centrifuged for 5 min. This washing step was carried out a second time. The flowthrough was discarded and followed by centrifugation for 5 min to  remove residual SEWS-M buffer. Finally, 30 μl nuclease-free water were added to each well and left to incubate at room temperature for 3 min. Subsequent centrifugation for 5 min eluted the DNA into a clean PCR plate. The resulting DNA was used for v5v7 16S rRNA region amplification without prior adjustment of DNA concentrations.
\ No newline at end of file
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx
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-- 
GitLab


From 41cdbbe6c4d4f65d78cda7879416346047bcb661 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 15:43:26 +0100
Subject: [PATCH 33/35] 16S rRNA prep protocol, assay

---
 .gitattributes                                   |   1 +
 assays/DNA Isolation/README.md                   |   1 +
 .../README.md                                    |   0
 .../dataset/.gitkeep                             |   0
 .../isa.assay.xlsx                               | Bin 0 -> 9837 bytes
 .../protocols/.gitkeep                           |   0
 .../LibraryPreparation16SrRNAProfiling.md        |   3 +++
 assays/MicrobiotaReconstitutionFlowpot/README.md |   1 +
 .../README.md                                    |   1 +
 9 files changed, 7 insertions(+)
 create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/README.md
 create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep
 create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx
 create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep
 create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md

diff --git a/.gitattributes b/.gitattributes
index eac0854..4b96006 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -14,3 +14,4 @@ assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Mono-association[[:space:]]experiment[[:space:]]of[[:space:]]R401[[:space:]]on[[:space:]]Athaliana[[:space:]]seedlings[[:space:]]in[[:space:]]agar[[:space:]]plates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Library[[:space:]]preparation[[:space:]]for[[:space:]]bacterial[[:space:]]16S[[:space:]]rRNA[[:space:]]gene[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/DNA Isolation/README.md b/assays/DNA Isolation/README.md
index e69de29..0519ecb 100644
--- a/assays/DNA Isolation/README.md	
+++ b/assays/DNA Isolation/README.md	
@@ -0,0 +1 @@
+ 
\ No newline at end of file
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/README.md b/assays/Library preparation for bacterial 16S rRNA gene profiling/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep b/assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx b/assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx
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diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md
new file mode 100644
index 0000000..17be9b0
--- /dev/null
+++ b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md	
@@ -0,0 +1,3 @@
+## Library preparation for bacterial 16S rRNA gene profiling
+
+The v5v7 variable regions of the bacterial *16S* rRNA gene were amplified in 96-wells plates through PCR using 799F and 1192R primers (PCR I; **Table S4**). PCR I was performed with 0.4 μl DFS-Taq polymerase (BIORON) in 25-μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM 799F, 0.75 μl 10 μM 1192R, 1 μl of isolated DNA and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 25 cycles of 94°C for 30 s, 55°C for 30 s,72°C for 1 min, then a final extension at 72 °C for 10 min. Remaining primers and dNTPs were digested by Antarctic phosphatase and Exonuclease I. To each 25 μl PCR reaction, 1 μl of Antarctic phosphatase (New-England BioLabs), 1 μl of Exonuclease I (New-England BioLabs) and 3 μl of Antarctic phosphatase buffer (New-England BioLabs) were added; the resulting mixture was incubated at 37 °C for 30 min followed by heat inactivation of the enzymes at 85 °C for 15 min. The digested PCR I product was centrifuged at 3,000 rpm and 4 °C for 10 minutes. Then, 3 μl of the supernatant were used as a template in a second PCR, using a unique combination of uniquely barcoded 799F- and 1192-based primers containing Illumina adaptors for each sample (PCR II; **Table S4**). PCR II was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions containing 2.5 μl 10x incomplete buffer, 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM unique forward primer, 0.75 μl 10 μM unique reverse primer, 3 μl cleaned PCR I-product and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 10 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, then a final extension at 72 °C for 10 min. Subsequently, 5 μl of the PCR product were combined with 1 ml Orange DNA Loading Dye, loaded on a 1% agarose gel containing 0.05% EtBr, and run at 110 mV. The expected PCR-product was an approx. 500 bp band containing the variable v5v7 regions, barcodes, and Illumina adaptors. After verification of successful amplification, samples were purified by AMPure XP (Beckman-Coulter) according to the manufacturer’s protocol. The DNA concentrations of each sample were quantified using the Quant-iT dsDNA Assay-Kit (Invitrogen) and pooled per full factorial replicate in equimolar values. All pools were purified twice using AMPure XP and fluorescently quantified using the Quant-iT dsDNA Assay-Kit. All pooled samples were combined into a single pool based on equimolarity and subsequently purified three times using AMPure XP and fluorescently quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). The final concentration was set to 10 ng/μl. Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers (**Table S4**).
diff --git a/assays/MicrobiotaReconstitutionFlowpot/README.md b/assays/MicrobiotaReconstitutionFlowpot/README.md
index e69de29..0519ecb 100644
--- a/assays/MicrobiotaReconstitutionFlowpot/README.md
+++ b/assays/MicrobiotaReconstitutionFlowpot/README.md
@@ -0,0 +1 @@
+ 
\ No newline at end of file
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
index e69de29..0519ecb 100644
--- a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md	
+++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md	
@@ -0,0 +1 @@
+ 
\ No newline at end of file
-- 
GitLab


From 7be4314fe65e651562401f735942613dfdc367e7 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 16:20:48 +0100
Subject: [PATCH 34/35] 16S profiling analysis

---
 .gitattributes                                  |   2 ++
 assays/Analysis of 16S profiling/README.md      |   0
 .../Analysis of 16S profiling/dataset/.gitkeep  |   0
 .../dataset/ComProfiling.jpg                    |   3 +++
 assays/Analysis of 16S profiling/isa.assay.xlsx | Bin 0 -> 11322 bytes
 .../protocols/.gitkeep                          |   0
 .../protocols/16SProfilingAnalysis.md           |  12 ++++++++++++
 7 files changed, 17 insertions(+)
 create mode 100644 assays/Analysis of 16S profiling/README.md
 create mode 100644 assays/Analysis of 16S profiling/dataset/.gitkeep
 create mode 100644 assays/Analysis of 16S profiling/dataset/ComProfiling.jpg
 create mode 100644 assays/Analysis of 16S profiling/isa.assay.xlsx
 create mode 100644 assays/Analysis of 16S profiling/protocols/.gitkeep
 create mode 100644 assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md

diff --git a/.gitattributes b/.gitattributes
index 4b96006..7bc447f 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -15,3 +15,5 @@ assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merg
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 assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Library[[:space:]]preparation[[:space:]]for[[:space:]]bacterial[[:space:]]16S[[:space:]]rRNA[[:space:]]gene[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/ComProfiling.jpg filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Analysis of 16S profiling/README.md b/assays/Analysis of 16S profiling/README.md
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diff --git a/assays/Analysis of 16S profiling/dataset/.gitkeep b/assays/Analysis of 16S profiling/dataset/.gitkeep
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diff --git a/assays/Analysis of 16S profiling/dataset/ComProfiling.jpg b/assays/Analysis of 16S profiling/dataset/ComProfiling.jpg
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diff --git a/assays/Analysis of 16S profiling/protocols/.gitkeep b/assays/Analysis of 16S profiling/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md b/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md
new file mode 100644
index 0000000..19f9193
--- /dev/null
+++ b/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md	
@@ -0,0 +1,12 @@
+## Analysis of 16S profiling data
+
+### ASV table generation
+
+Amplicon sequencing reads from *A. thaliana* roots and Flowpot soil were quality filtered and demultiplexed according to their barcode sequence using QIIME (26) and unique amplicon sequencing variants (ASVs) were inferred from error-corrected reads, followed by chimera filtering. ASVs were mapped to the reference 16S rRNA sequences (downloaded from “www.at-sphere.com”) to generate an ASV count table. All steps were carried out using the Rbec R package (27). Analysis was performed on samples with a sequencing depth of at least 500 high-quality reads.
+
+
+### Alpha- and beta-diversity
+
+Analyses and visualization were performed in the R statistical environment (Version 4.1.2).
+Alpha and beta diversity were calculated on non-rarefied ASV count tables (28). Alpha-diversity (Shannon index) was calculated with the “plot_richness” function in phyloseq package (29).
+Beta-diversity (Bray-Curtis dissimilarities) was calculated using the “ordinate” function in phyloseq package and used for unconstrained ordination by Principal Coordinate Analysis (PCoA). Statistical significances were assessed using permutational multi-variate analysis of variance (PERMANOVA) using the adonis function in the vegan package.
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From 8912854314db1ba65cb4cbbb79e7810113a4b479 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 16:30:52 +0100
Subject: [PATCH 35/35] figure descr

---
 .gitattributes                                           | 1 +
 assays/Analysis of 16S profiling/dataset/ComProfiling.md | 3 +++
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 create mode 100644 assays/Analysis of 16S profiling/dataset/ComProfiling.md

diff --git a/.gitattributes b/.gitattributes
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@@ -17,3 +17,4 @@ assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -t
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 assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/ComProfiling.jpg filter=lfs diff=lfs merge=lfs -text
+assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/ComProfiling.md filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/Analysis of 16S profiling/dataset/ComProfiling.md b/assays/Analysis of 16S profiling/dataset/ComProfiling.md
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+version https://git-lfs.github.com/spec/v1
+oid sha256:922853bf9051d5889e00ee8abecfb4a2f9d775bdc2260eb08105c68b6a75cfe9
+size 2429
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