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] 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
new file mode 100644
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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
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