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] 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
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diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx
index 69890d3be81d994fb8a52d2a22db9d79c3b0b62c..e846156dd260e8768b142f4e0c7d8c94e6b7ff3c 100644
GIT binary patch
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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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/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
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diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx
index ba6e39478168c92e5be36ba5756b6f5079d10710..b15f1a4b4e624c8fb1fa381bd79da504ac15d5de 100644
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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
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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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GIT binary patch
delta 822
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-- 
GitLab