From b9d926a2d53addb200356925af41fbd63339fa1b Mon Sep 17 00:00:00 2001 From: Sabrina Zander <sabrina.zander@uni-duesseldorf.de> Date: Tue, 21 Jan 2025 09:24:56 +0100 Subject: [PATCH] add study1_tag integration --- studies/1_TagIntegration/README.md | 1 + studies/1_TagIntegration/isa.study.xlsx | Bin 0 -> 10594 bytes studies/1_TagIntegration/protocols/.gitkeep | 0 .../protocols/Tag integration.md | 2 ++ studies/1_TagIntegration/resources/.gitkeep | 0 .../20201202_Tagging vectors dtxR.xlsx | Bin 0 -> 20117 bytes .../resources/DtxR-His-C-2link.cm5 | Bin 0 -> 4204 bytes .../resources/DtxR-Strep-N-4link.cm5 | Bin 0 -> 3578 bytes 8 files changed, 3 insertions(+) create mode 100644 studies/1_TagIntegration/README.md create mode 100644 studies/1_TagIntegration/isa.study.xlsx create mode 100644 studies/1_TagIntegration/protocols/.gitkeep create mode 100644 studies/1_TagIntegration/protocols/Tag integration.md create mode 100644 studies/1_TagIntegration/resources/.gitkeep create mode 100644 studies/1_TagIntegration/resources/20201202_Tagging vectors dtxR.xlsx create mode 100644 studies/1_TagIntegration/resources/DtxR-His-C-2link.cm5 create mode 100644 studies/1_TagIntegration/resources/DtxR-Strep-N-4link.cm5 diff --git a/studies/1_TagIntegration/README.md b/studies/1_TagIntegration/README.md new file mode 100644 index 0000000..20014f9 --- /dev/null +++ b/studies/1_TagIntegration/README.md @@ -0,0 +1 @@ +Every possible combination was constructed (N-terminal, C-terminal, His, Strep, long linker (4x), short linker (2x)) in every possible combination in pK19-plasmids. 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a/studies/1_TagIntegration/protocols/.gitkeep b/studies/1_TagIntegration/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/studies/1_TagIntegration/protocols/Tag integration.md b/studies/1_TagIntegration/protocols/Tag integration.md new file mode 100644 index 0000000..8fda6f7 --- /dev/null +++ b/studies/1_TagIntegration/protocols/Tag integration.md @@ -0,0 +1,2 @@ +Plasmids were constructed by enzymatically assembling the generated DNA fragments into a cut vector backbone using Gibson assembly (Gibson et al., 2009). Sequencing of the final plasmid was performed by Eurofins Genomics (Ebersberg, Germany). +For the integration of the His-Tag and a linker (GGGS2) at the C- or N-terminus of DtxR in the genome of C. glutamicum, the suicide vector pK19-mobsacB was used (Schäfer et al., 1994). Electrocompetent C. glutamicum cells were transformed with the isolated plasmid by electroporation (van der Rest et al., 1999). Then, the first and second recombination events were performed and verified as described in previous studies (Niebisch and Bott, 2001). 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