diff --git a/assays/BGC prediction using antiSMASH/isa.assay.xlsx b/assays/BGC prediction using antiSMASH/isa.assay.xlsx index b06b8354d92b038d824c16bc5ed5745d5632e109..0c87e3146728ed2f1778c0a9d98276d12929de68 100644 Binary files a/assays/BGC prediction using antiSMASH/isa.assay.xlsx and b/assays/BGC prediction using antiSMASH/isa.assay.xlsx differ diff --git a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx index e846156dd260e8768b142f4e0c7d8c94e6b7ff3c..13af4a103f05c97a1988a354cd472f29773efa4d 100644 Binary files a/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx and b/assays/DetectionOfR401DAPGandPyoverdine/isa.assay.xlsx differ diff --git a/assays/Metabolomics Analysis/isa.assay.xlsx b/assays/Metabolomics Analysis/isa.assay.xlsx index c876c4ee3472462539cb9506cce354ad57473dfa..2828a44d204d086e4ac9ffa61342b8a760a97d20 100644 Binary files a/assays/Metabolomics Analysis/isa.assay.xlsx and b/assays/Metabolomics Analysis/isa.assay.xlsx differ diff --git a/assays/Mutant Generation/isa.assay.xlsx b/assays/Mutant Generation/isa.assay.xlsx index 0d4d9bfb148129fe76665a291dfab96bad264f67..72a950918f71def66c69eec60ba96bd489ace3a9 100644 Binary files a/assays/Mutant Generation/isa.assay.xlsx and b/assays/Mutant Generation/isa.assay.xlsx differ diff --git a/assays/Mutant Generation/protocols/R401andEcoliConjugation.md b/assays/Mutant Generation/protocols/R401andEcoliConjugation.md new file mode 100644 index 0000000000000000000000000000000000000000..75c4ee8e2e563494218c0708c6a56abbd051da6f --- /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 0000000000000000000000000000000000000000..dcfd4b1b9eea8898d30717fa50bd3ca431d687d6 --- /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 index c5fbcc9dda8928f7c4f7b86087023549c3be61da..bc01a83524cbc783648a199c29b01b6525d91d5a 100644 Binary files a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx and b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index b15f1a4b4e624c8fb1fa381bd79da504ac15d5de..a5d23c084a7cfa655626c822b84756d28b190a29 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx index f79bce8581a85dd69170620fa7fb175e0bed438c..725e55d80bc97a6a08547243fe24be57e8c0e833 100644 Binary files a/studies/Bacterial culture conditions/isa.study.xlsx and b/studies/Bacterial culture conditions/isa.study.xlsx differ diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx index 36f4e75c4a78fa0fdeaa5b15460c17377974b9a2..0ea160aee9616d65c884afa9f8da53d84478e394 100644 Binary files a/studies/Plant Growth Conditions/isa.study.xlsx and b/studies/Plant Growth Conditions/isa.study.xlsx differ