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[Abstract](## Abstract) +2. [Studies](## Studies) +3. [Assays](## Assays) +4. [Licence](## License) + + + +## Abstract + +Eukaryotic life has been shaped fundamentally by the integration of bacterial endosymbionts. The trypanosomatid *Angomonas deanei* that contains a beta-proteobacterial endosymbiont, represents an emerging model to elucidate initial steps in symbiont integration. Although the repertoire of genetic tools for *A. deanei* is growing, no conditional gene expression system is available yet, which would be key for the functional characterization of essential or expression of toxic proteins. Development of a conditional expression system based on endogenous RNA polymerase II (POLII) is hampered by the absence of information on transcription signals in *A. deanei* as well as the unusual genetic system used in the Trypanosomatidae that relies on read-through transcription. This mode of transcription can result in polar effects when manipulating expression of genes in their endogenous loci. Finally, only few resistance markers are available for *A. deanei* yet, restricting the number of genetic modifications that can be introduced into one strain. To increase the range of possible genetic manipulations in *A. deanei*, and in particular, build the base for a conditional expression system that does not interfere with the endogenous gene expression machinery, here we (i) implemented two new drug resistance markers, (ii) identified the spacer upstream of the rDNA array on chromosome 13 as transcriptionally silent genomic locus, and (iii) used this locus for engineering an ectopic expression system that depends on the T7 RNA polymerase expressed from the delta-amastin locus. We show that transgene expression in this system is independent of the activity of endogenous RNA polymerases, reaches expression levels similar to the previously described POLII-dependent expression from the gamma-amastin locus, and can be applied for studying endosymbiosis. In sum, the new tools expand the possibilities for genetic manipulations of *A. deanei* and provide a solid base for the development of an ectopic conditional expression system. + +## Studies + +The Studies (S) are named after the names of the chapters in the publication. +* S1_Identification of potentially silent loci +* S2_Heterologous expression of the T7RNAP +* S3_Implementation of new antibiotic resistance cassettes +* S4_T7RNAP-driven expression from the rDNA spacer +* S5_Analysis of gene expression strength + +## Assays + +The [Assays] folder contains the results of the individual experiments. The raw and processed data are stored in the `dataset` folder of the assay and the corresponding protocols are in the `protocol` folder. + +### Chapter 1: Identification of potentially silent loci +* S1_1_rDNA spacers on different chromosomes // **Fig. 1** + +### Chapter 2: Heterologous expression of the T7RNAP +* S2_1_Genomic map of Adea319 // **Fig. 2A** +* S2_2_Verification of genomic integration by PCR // **Fig. 2B** +* S2_3_Verification of T7RNAP production by Western Blot // **Fig. 2C** + +### Chapter 3: Implementation of new antibiotic resistance cassettes +* S3_1_Growth curve of WT and Adea373 on blast // **Fig. 3A** +* S3_2_Growth curve of WT and Adea384 on nours // **Fig. 3B** + +### Chapter 4: T7RNAP-driven expression from the rDNA spacer +* S4_1_Genomic map of Adea400 // **Fig. 4A** +* S4_2_Verification of insertion by PCR I // **Fig. 4B** +* S4_3_Verification of insertion by PCR II // **Fig. 4C** +* S4_4_Verification of insertion by Southern Blot // **Fig. 4D** +* S4_5_Growth curves of Adea319 and Adea400 // **Fig. 4E** +* S4_6_Knock-out strategy for T7RNAP // **Fig. 4F** +* S4_7_Verification of knock-out by PCR // **Fig. 4G** +* S4_5_Growth curves of WT, Adea373 and Adea442 // **Fig. 4H** + +### Chapter 5: Analysis of gene expression strength +* S5_1_Genomic map of Adea456 // **Fig. 5A** +* S5_2_Verification of insertion by Southern Blot // **Fig. 5B** +* S5_3_Growth curves of WT and Adea456 // **Fig. 5C** +* S5_4_Epifluorescence microscopy of Adea126 and Adea456 // **Fig. 5D** +* S5_5_Quantification of fluorescent cells // **Fig. 5E** +* S5_6_In gel fluorescence assay of Adea126 and Adea456 // **Fig. 5F (top)** +======= +# T7 RNA polymerase-based gene expression from a transcriptionally silent rDNA spacer in the endosymbiont-harboring trypanosomatid *Angomonas deanei* + +This ARC [Annoteted Research Context](https://arc-rdm.org/) contains the original data of the Kröninger et al. publication. + +## Original Publication + +Publication +doi + +## Table of Contents + +1. [Abstract](## Abstract) +2. [Studies](## Studies) +3. [Assays](## Assays) +4. [Licence](## License) + + + +## Abstract + +Eukaryotic life has been shaped fundamentally by the integration of bacterial endosymbionts. The trypanosomatid *Angomonas deanei* that contains a beta-proteobacterial endosymbiont, represents an emerging model to elucidate initial steps in symbiont integration. Although the repertoire of genetic tools for *A. deanei* is growing, no conditional gene expression system is available yet, which would be key for the functional characterization of essential or expression of toxic proteins. Development of a conditional expression system based on endogenous RNA polymerase II (POLII) is hampered by the absence of information on transcription signals in *A. deanei* as well as the unusual genetic system used in the Trypanosomatidae that relies on read-through transcription. This mode of transcription can result in polar effects when manipulating expression of genes in their endogenous loci. Finally, only few resistance markers are available for *A. deanei* yet, restricting the number of genetic modifications that can be introduced into one strain. To increase the range of possible genetic manipulations in *A. deanei*, and in particular, build the base for a conditional expression system that does not interfere with the endogenous gene expression machinery, here we (i) implemented two new drug resistance markers, (ii) identified the spacer upstream of the rDNA array on chromosome 13 as transcriptionally silent genomic locus, and (iii) used this locus for engineering an ectopic expression system that depends on the T7 RNA polymerase expressed from the delta-amastin locus. We show that transgene expression in this system is independent of the activity of endogenous RNA polymerases, reaches expression levels similar to the previously described POLII-dependent expression from the gamma-amastin locus, and can be applied for studying endosymbiosis. In sum, the new tools expand the possibilities for genetic manipulations of *A. deanei* and provide a solid base for the development of an ectopic conditional expression system. + +## Studies + +The Studies (S) are named after the names of the chapters in the publication. +* S1_Identification of potentially silent loci +* S2_Heterologous expression of the T7RNAP +* S3_Implementation of new antibiotic resistance cassettes +* S4_T7RNAP-driven expression from the rDNA spacer +* S5_Analysis of gene expression strength + +## Assays + +The [Assays] folder contains the results of the individual experiments. The raw and processed data are stored in the `dataset` folder of the assay and the corresponding protocols are in the `protocol` folder. + +### Chapter 1: Identification of potentially silent loci +* S1_1_rDNA spacers on different chromosomes // **Fig. 1** + +### Chapter 2: Heterologous expression of the T7RNAP +* S2_1_Genomic map of Adea319 // **Fig. 2A** +* S2_2_Verification of genomic integration by PCR // **Fig. 2B** +* S2_3_Verification of T7RNAP production by Western Blot // **Fig. 2C** + +### Chapter 3: Implementation of new antibiotic resistance cassettes +* S3_1_Growth curve of WT and Adea373 on blast // **Fig. 3A** +* S3_2_Growth curve of WT and Adea384 on nours // **Fig. 3B** + +### Chapter 4: T7RNAP-driven expression from the rDNA spacer +* S4_1_Genomic map of Adea400 // **Fig. 4A** +* S4_2_Verification of insertion by PCR I // **Fig. 4B** +* S4_3_Verification of insertion by PCR II // **Fig. 4C** +* S4_4_Verification of insertion by Southern Blot // **Fig. 4D** +* S4_5_Growth curves of Adea319 and Adea400 // **Fig. 4E** +* S4_6_Knock-out strategy for T7RNAP // **Fig. 4F** +* S4_7_Verification of knock-out by PCR // **Fig. 4G** +* S4_5_Growth curves of WT, Adea373 and Adea442 // **Fig. 4H** + +### Chapter 5: Analysis of gene expression strength +* S5_1_Genomic map of Adea456 // **Fig. 5A** +* S5_2_Verification of insertion by Southern Blot // **Fig. 5B** +* S5_3_Growth curves of WT and Adea456 // **Fig. 5C** +* S5_4_Epifluorescence microscopy of Adea126 and Adea456 // **Fig. 5D** +* S5_5_Quantification of fluorescent cells // **Fig. 5E** +* S5_6_In gel fluorescence assay of Adea126 and Adea456 // **Fig. 5F (top)** +>>>>>>> 90829c7 (Created my first ARC and added data) +* S5_7_Quantification of in gel assay // **Fig. 5F (bottom)** \ No newline at end of file diff --git a/assays/S1_1_rDNA-spacers-on-different-chrosomosomes/README.md b/assays/S1_1_rDNA-spacers-on-different-chrosomosomes/README.md new file mode 100644 index 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--git a/assays/S2_1_Genomic-map-of-Adea319/isa.assay.xlsx b/assays/S2_1_Genomic-map-of-Adea319/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..e39f4a60a3435989fbe395a05143320ffdd9316e Binary files /dev/null and b/assays/S2_1_Genomic-map-of-Adea319/isa.assay.xlsx differ diff --git a/assays/S2_1_Genomic-map-of-Adea319/protocols/.gitkeep b/assays/S2_1_Genomic-map-of-Adea319/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_1_Genomic-map-of-Adea319/protocols/Protocol_Cloning.md b/assays/S2_1_Genomic-map-of-Adea319/protocols/Protocol_Cloning.md new file mode 100644 index 0000000000000000000000000000000000000000..6d37c3acb20497d30c723910c39a82d5b3b6cd15 --- /dev/null +++ b/assays/S2_1_Genomic-map-of-Adea319/protocols/Protocol_Cloning.md @@ -0,0 +1,11 @@ +# Generation of plasmids (cloning) + +* All plasmids for this study were products of Gibson [Gibson et al. 2009] or Golden Gate [Engler et al. 2008] assemblies as described previously [Morales et al. 2023]. +* The cloning fragments, which were needed for the assemblies, were generated by PCR using the Phusion polymerase (New England Biolabs). Primers and templates are listed in S2 Table. +* The correct sizes of cloning fragments were verified by electrophoresis. +* PCR products matching the expected sizes were excised from the gel and purified using the Monarch DNA Gel Extraction kit (New England Biolabs). The concentration of the DNA was determined using the NanoPhotometer NP80 (Implen). +* The molar concentration of the insert(s) usually exceeded the vector backbone concentration by a factor of 3. +* After the assembly, 1 µl of DpnI was added to each assembly mix to remove residual PCR template present in the mix. +* Then, E. coli TOP10 cells were transformed with the assembly mix and recombinant cells selected on lysogeny broth (LB) plates containing 100 µg/ml ampicillin. +* Recovered clones were inoculated in 5 ml LB liquid medium with ampicillin (see above) and used for plasmid purification (Monarch Plasmid Miniprep kit, New England Biolabs). +* Plasmids were verified by control digests and sequencing (Microsynth). \ No newline at end of file diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/README.md b/assays/S2_2_Verification-of-genomic-integration-by-PCR/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/dataset/.gitkeep b/assays/S2_2_Verification-of-genomic-integration-by-PCR/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/dataset/2023-11-24 SYBR Safe stained agarose gel.tif b/assays/S2_2_Verification-of-genomic-integration-by-PCR/dataset/2023-11-24 SYBR Safe stained agarose gel.tif new file mode 100644 index 0000000000000000000000000000000000000000..10168fad2e5c740699458ab6ef5911b0d6300f48 --- /dev/null +++ b/assays/S2_2_Verification-of-genomic-integration-by-PCR/dataset/2023-11-24 SYBR Safe stained agarose gel.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d133cb69eb9d7032e57d55eb711a8123e1d3948061be2efeb9060e7af9d8d2c3 +size 308052 diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/isa.assay.xlsx b/assays/S2_2_Verification-of-genomic-integration-by-PCR/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..8737bff2834df247fe2f815a23c4518df9325e38 Binary files /dev/null and b/assays/S2_2_Verification-of-genomic-integration-by-PCR/isa.assay.xlsx differ diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/protocols/.gitkeep b/assays/S2_2_Verification-of-genomic-integration-by-PCR/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_2_Verification-of-genomic-integration-by-PCR/protocols/Protocol_PCR.md b/assays/S2_2_Verification-of-genomic-integration-by-PCR/protocols/Protocol_PCR.md new file mode 100644 index 0000000000000000000000000000000000000000..832b0c391c14cff7c9aa7c6af50a487a7296ac84 --- /dev/null +++ b/assays/S2_2_Verification-of-genomic-integration-by-PCR/protocols/Protocol_PCR.md @@ -0,0 +1,9 @@ +# Polymerase chain reaction (PCR) + +* All PCR reactions contained 100 ng for plasmid DNA or 50 ng of genomic DNA. +* For the amplification the Phusion polymerase (New England Biolabs) was used according to the manufacturer's instructions. +* The primer concentrations equaled 0.5 µM per primer. +* All reactions contained 1.25 M betaine as PCR enhancer. +* PCR steps: Denaturation at 95 °C for 20 s per cycle, Annealing at primer specific temperature for 20 s per cycle, elongation at 72 °C for 30 s per 1 kb +* Number of cycles: 30 +* TD-PCR for verification of genomic insertion (in delta-amastin locus): 72-68 °C annealing temperature for 2 cycles each, followed by 25 cycles at 60 °C, elongation time: 4 min \ No newline at end of file diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/README.md b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/.gitkeep b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-07 Stain Free Gel.jpg b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-07 Stain Free Gel.jpg new file mode 100644 index 0000000000000000000000000000000000000000..5eadf4430147381ff879ff7747a6944be8007425 --- /dev/null +++ b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-07 Stain Free Gel.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8c5915c395cae1a1fbc70ebbc5e8992a866e33845a7b13cfc841cc13b0b2da1f +size 60940 diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-11 Chemiluminescence + Colorimetric.jpg b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-11 Chemiluminescence + Colorimetric.jpg new file mode 100644 index 0000000000000000000000000000000000000000..d68057f2e649f9be4255b0a4263ad6d980344557 --- /dev/null +++ b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/dataset/2023-07-11 Chemiluminescence + Colorimetric.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1dcf2f12ca285345ba823d7281424b53632994e7400c7215fa8e828c92dc874c +size 33269 diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/isa.assay.xlsx b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..081a20236189a090e65b61e116d300ed06a10cf0 Binary files /dev/null and b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/isa.assay.xlsx differ diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/protocols/.gitkeep b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/protocols/Protocol_Western Blot.md b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/protocols/Protocol_Western Blot.md new file mode 100644 index 0000000000000000000000000000000000000000..ee3bc3a56d781ba4ec69afa28a67f89cb50e43cc --- /dev/null +++ b/assays/S2_3_Verification-of-T7RNAP-production-by-Western-Blot/protocols/Protocol_Western Blot.md @@ -0,0 +1,12 @@ +# SDS-PAGE & Western Blot + +* For the detection of specific proteins, we grew 10 ml cultures of the relevant strains to mid-exponential phase. +* We harvested the cells by centrifugation and precipitated the proteins using trichloroacetic acid [Morales et al. 2023]. +* The precipitate was resuspended in urea buffer (8 M urea, 100 mM Tris, 5.25 mM EDTA, pH 8.6) and the protein concentration was determined using Pierce 660nm Protein Assay Reagent (ThermoFisher) and the FLUOstar Omega Plate Reader (BMG Labtech). A bovine serum albumin (BSA) standard curved served as reference. +* For each sample, 25 µg protein were mixed with Lämmli sample buffer (final concentrations: 63 mM Tris-HCl, pH 6.8, 10 mM dithiothreitol, 10% v/v glycerol, 2% SDS w/v, and 0.0025% w/v bromophenol blue), incubated for 5 min at 95 °C, and loaded onto a 10 % SDS polyacrylamid gel containing 0.5 % TCE. +* PAGE and protein blot to a PVDF membrane (Amersham Hybond, 0.45 nm, GE HealthCare Life Science) were performed as described previously [Morales et al. 2023]. +* After blotting, proteins were detected immunologically. In brief, the membrane was blocked with 5 % w/v milk powder for 1 h at room temperature (or 24 h at 4 °C) and washed three times for 5 min with 1x PBST (phosphate-buffered saline including 0.05 % v/v Tween-20) before the primary antibody was added. +* For detection of T7RNAP, an α-T7RNAP antibody (29943-1-AP, Proteintech) was used in a 1:5000 dilution overnight at 4 °C. Before the secondary conjugate was added, the membrane was washed again three times for 5 min with 1x PBST. +* The α-T7RNAP antibody was detected using α-rabbit IgG HRP conjugate (7074, CellSignaling), which was applied for 1 h at room temperature followed by three more washing steps (see above). +* Chemiluminescence was started by the addition of SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) and it was recorded using the ChemiDoc MP (Biorad). +* The TCE staining was visualized using the "stain-free gel" option of the ChemiDoc MP (Biorad). \ No newline at end of file diff --git a/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/README.md b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/dataset/.gitkeep b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/dataset/.gitkeep new 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0000000000000000000000000000000000000000..82d10a56452b59dc911ec69b6ccb237ea7edfaf5 Binary files /dev/null and b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/dataset/Growth curve_Adea373 and WT on blast.xlsx differ diff --git a/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/isa.assay.xlsx b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..4f5b716247a5005d1c5e61b7a162252f2357e5e4 Binary files /dev/null and b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/isa.assay.xlsx differ diff --git a/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/protocols/.gitkeep b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/protocols/Protocol_Growth curve.md b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/protocols/Protocol_Growth curve.md new file mode 100644 index 0000000000000000000000000000000000000000..e0ae8689cd88ed3c5363ee2bbf273cb4dd203c1b --- /dev/null +++ b/assays/S3_1_Growth-curve-of-WT-and-Adea373-on-blast/protocols/Protocol_Growth curve.md @@ -0,0 +1,12 @@ +# Growth curves + +* For the analysis of the growth behavior of different A. deanei strains, precultures were grown to mid-exponential phase (approximately 1-5 x 10^7 cells/ml) with all selective drugs that would allow growth of the specific strains. +* Then, three replicates were prepared with 5 ml BHI plus hemin and antibiotics as indicated below and inoculated to an initial cell density of 1 x 10^5 cells/ml. +* The tubes were incubated at 28 °C without agitation and the cell density was measured every 20-24 h using the MultiSizer 4e (Beckman Coulter). + +## Antibiotic concentrations (final concentrations): + +* Neomycin (G418, Sigma-Aldrich): 500 µg/ml +* Hygromycin B (Invivogen): 500 µg/ml +* Blasticidin (Invivogen): 75 µg/ml +* Nourseothricin (Carl Roth): 200 µg/ml \ No newline at end of file diff --git a/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/README.md b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/dataset/.gitkeep b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/dataset/Growth curve_Adea384 and WT on nours.pzf b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/dataset/Growth curve_Adea384 and WT on nours.pzf new file mode 100644 index 0000000000000000000000000000000000000000..dda64698650787818ca104127a62dce1cbf9e3e9 --- /dev/null +++ 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/dev/null and b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/isa.assay.xlsx differ diff --git a/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/protocols/.gitkeep b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/protocols/Protocol_Growth curve.md b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/protocols/Protocol_Growth curve.md new file mode 100644 index 0000000000000000000000000000000000000000..e0ae8689cd88ed3c5363ee2bbf273cb4dd203c1b --- /dev/null +++ b/assays/S3_2_Growth-curve-of-WT-and-Adea384-on-nours/protocols/Protocol_Growth curve.md @@ -0,0 +1,12 @@ +# Growth curves + +* For the analysis of the growth behavior of different A. deanei strains, precultures were grown to mid-exponential phase (approximately 1-5 x 10^7 cells/ml) with all selective drugs that would allow growth of the specific strains. +* Then, three replicates were prepared with 5 ml BHI plus hemin and antibiotics as indicated below and inoculated to an initial cell density of 1 x 10^5 cells/ml. +* The tubes were incubated at 28 °C without agitation and the cell density was measured every 20-24 h using the MultiSizer 4e (Beckman Coulter). + +## Antibiotic concentrations (final concentrations): + +* Neomycin (G418, Sigma-Aldrich): 500 µg/ml +* Hygromycin B (Invivogen): 500 µg/ml +* Blasticidin (Invivogen): 75 µg/ml +* Nourseothricin (Carl Roth): 200 µg/ml \ No newline at end of file diff --git a/assays/S3_3_Genomic-maps-of-Adea373-and-Adea384/README.md b/assays/S3_3_Genomic-maps-of-Adea373-and-Adea384/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S3_3_Genomic-maps-of-Adea373-and-Adea384/dataset/.gitkeep b/assays/S3_3_Genomic-maps-of-Adea373-and-Adea384/dataset/.gitkeep new file mode 100644 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b/assays/S3_3_Genomic-maps-of-Adea373-and-Adea384/protocols/Protocol_Cloning.md @@ -0,0 +1,11 @@ +# Generation of plasmids (cloning) + +* All plasmids for this study were products of Gibson [Gibson et al. 2009] or Golden Gate [Engler et al. 2008] assemblies as described previously [Morales et al. 2023]. +* The cloning fragments, which were needed for the assemblies, were generated by PCR using the Phusion polymerase (New England Biolabs). Primers and templates are listed in S2 Table. +* The correct sizes of cloning fragments were verified by electrophoresis. +* PCR products matching the expected sizes were excised from the gel and purified using the Monarch DNA Gel Extraction kit (New England Biolabs). The concentration of the DNA was determined using the NanoPhotometer NP80 (Implen). +* The molar concentration of the insert(s) usually exceeded the vector backbone concentration by a factor of 3. +* After the assembly, 1 µl of DpnI was added to each assembly mix to remove residual PCR template present in the mix. +* Then, E. coli TOP10 cells were transformed with the assembly mix and recombinant cells selected on lysogeny broth (LB) plates containing 100 µg/ml ampicillin. +* Recovered clones were inoculated in 5 ml LB liquid medium with ampicillin (see above) and used for plasmid purification (Monarch Plasmid Miniprep kit, New England Biolabs). +* Plasmids were verified by control digests and sequencing (Microsynth). \ No newline at end of file diff --git a/assays/S4_1_Genomic-map-of-Adea400/README.md b/assays/S4_1_Genomic-map-of-Adea400/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_1_Genomic-map-of-Adea400/dataset/.gitkeep b/assays/S4_1_Genomic-map-of-Adea400/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_1_Genomic-map-of-Adea400/isa.assay.xlsx b/assays/S4_1_Genomic-map-of-Adea400/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..4a56cabd7d4f073db8b102c849c5526ce4125dc4 Binary files /dev/null and b/assays/S4_1_Genomic-map-of-Adea400/isa.assay.xlsx differ diff --git a/assays/S4_1_Genomic-map-of-Adea400/protocols/.gitkeep b/assays/S4_1_Genomic-map-of-Adea400/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_1_Genomic-map-of-Adea400/protocols/Protocol_Cloning.md b/assays/S4_1_Genomic-map-of-Adea400/protocols/Protocol_Cloning.md new file mode 100644 index 0000000000000000000000000000000000000000..6d37c3acb20497d30c723910c39a82d5b3b6cd15 --- /dev/null +++ b/assays/S4_1_Genomic-map-of-Adea400/protocols/Protocol_Cloning.md @@ -0,0 +1,11 @@ +# Generation of plasmids (cloning) + +* All plasmids for this study were products of Gibson [Gibson et al. 2009] or Golden Gate [Engler et al. 2008] assemblies as described previously [Morales et al. 2023]. +* The cloning fragments, which were needed for the assemblies, were generated by PCR using the Phusion polymerase (New England Biolabs). Primers and templates are listed in S2 Table. +* The correct sizes of cloning fragments were verified by electrophoresis. +* PCR products matching the expected sizes were excised from the gel and purified using the Monarch DNA Gel Extraction kit (New England Biolabs). The concentration of the DNA was determined using the NanoPhotometer NP80 (Implen). +* The molar concentration of the insert(s) usually exceeded the vector backbone concentration by a factor of 3. +* After the assembly, 1 µl of DpnI was added to each assembly mix to remove residual PCR template present in the mix. +* Then, E. coli TOP10 cells were transformed with the assembly mix and recombinant cells selected on lysogeny broth (LB) plates containing 100 µg/ml ampicillin. +* Recovered clones were inoculated in 5 ml LB liquid medium with ampicillin (see above) and used for plasmid purification (Monarch Plasmid Miniprep kit, New England Biolabs). +* Plasmids were verified by control digests and sequencing (Microsynth). \ No newline at end of file diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/README.md b/assays/S4_2_Verification-of-insertion-by-PCR-I/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/dataset/.gitkeep b/assays/S4_2_Verification-of-insertion-by-PCR-I/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/dataset/2023-06-13 SYBR Safe stained agarose gel.jpg b/assays/S4_2_Verification-of-insertion-by-PCR-I/dataset/2023-06-13 SYBR Safe stained agarose gel.jpg new file mode 100644 index 0000000000000000000000000000000000000000..617652b3e578769a6689a1522e0cb3e03eb2c499 --- /dev/null +++ b/assays/S4_2_Verification-of-insertion-by-PCR-I/dataset/2023-06-13 SYBR Safe stained agarose gel.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0a7e187c26e94dabd512f66a2bf9cf749f98a0e1e129bf728c7780c4bdd6178b +size 41192 diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/isa.assay.xlsx b/assays/S4_2_Verification-of-insertion-by-PCR-I/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..fbdb2452bec9235af41e312784dadbe786b7c081 Binary files /dev/null and b/assays/S4_2_Verification-of-insertion-by-PCR-I/isa.assay.xlsx differ diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/protocols/.gitkeep b/assays/S4_2_Verification-of-insertion-by-PCR-I/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_2_Verification-of-insertion-by-PCR-I/protocols/Protocol_PCR.md b/assays/S4_2_Verification-of-insertion-by-PCR-I/protocols/Protocol_PCR.md new file mode 100644 index 0000000000000000000000000000000000000000..df21ea168213193a8a1c160cad1f9fc88223ad16 --- /dev/null +++ b/assays/S4_2_Verification-of-insertion-by-PCR-I/protocols/Protocol_PCR.md @@ -0,0 +1,9 @@ +# Polymerase chain reaction + +* All PCR reactions contained 100 ng for plasmid DNA or 50 ng of genomic DNA. +* For the amplification the Phusion polymerase (New England Biolabs) was used according to the manufacturer's instructions. +* The primer concentrations equaled 0.5 µM per primer. +* All reactions contained 1.25 M betaine as PCR enhancer. +* PCR steps: Denaturation at 95 °C for 20 s per cycle, Annealing at primer specific temperature for 20 s per cycle, elongation at 72 °C for 30 s per 1 kb +* Number of cycles: 30 +* TD-PCR for verification of genomic insertion (in delta-amastin locus): 72-68 °C annealing temperature for 2 cycles each, followed by 25 cycles at 60 °C, elongation time: 4 min \ No newline at end of file diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/README.md b/assays/S4_3_Verification-of-insertion-by-PCR-II/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/dataset/.gitkeep b/assays/S4_3_Verification-of-insertion-by-PCR-II/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/dataset/2023-05-31 SYBR Safe stained agarose gel.jpg b/assays/S4_3_Verification-of-insertion-by-PCR-II/dataset/2023-05-31 SYBR Safe stained agarose gel.jpg new file mode 100644 index 0000000000000000000000000000000000000000..f97a099c99db6d316166660c1750e0f59228a886 --- /dev/null +++ b/assays/S4_3_Verification-of-insertion-by-PCR-II/dataset/2023-05-31 SYBR Safe stained agarose gel.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:76582ae12b1583a293f03dcbe7fb0bf79ab219ccd1a73bb0d25c8522db4f5da5 +size 44955 diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/isa.assay.xlsx b/assays/S4_3_Verification-of-insertion-by-PCR-II/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..5c3e253c12d562372446d1ae03c257b46561b5db Binary files /dev/null and b/assays/S4_3_Verification-of-insertion-by-PCR-II/isa.assay.xlsx differ diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/protocols/.gitkeep b/assays/S4_3_Verification-of-insertion-by-PCR-II/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_3_Verification-of-insertion-by-PCR-II/protocols/Protocol_PCR.md b/assays/S4_3_Verification-of-insertion-by-PCR-II/protocols/Protocol_PCR.md new file mode 100644 index 0000000000000000000000000000000000000000..df21ea168213193a8a1c160cad1f9fc88223ad16 --- /dev/null +++ b/assays/S4_3_Verification-of-insertion-by-PCR-II/protocols/Protocol_PCR.md @@ -0,0 +1,9 @@ +# Polymerase chain reaction + +* All PCR reactions contained 100 ng for plasmid DNA or 50 ng of genomic DNA. +* For the amplification the Phusion polymerase (New England Biolabs) was used according to the manufacturer's instructions. +* The primer concentrations equaled 0.5 µM per primer. +* All reactions contained 1.25 M betaine as PCR enhancer. +* PCR steps: Denaturation at 95 °C for 20 s per cycle, Annealing at primer specific temperature for 20 s per cycle, elongation at 72 °C for 30 s per 1 kb +* Number of cycles: 30 +* TD-PCR for verification of genomic insertion (in delta-amastin locus): 72-68 °C annealing temperature for 2 cycles each, followed by 25 cycles at 60 °C, elongation time: 4 min \ No newline at end of file diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/README.md b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/dataset/.gitkeep b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/dataset/2024-04-11 Chemiluminescence.tif b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/dataset/2024-04-11 Chemiluminescence.tif new file mode 100644 index 0000000000000000000000000000000000000000..219f57df229356fe893e90ef4feae9b1af44d031 --- /dev/null +++ b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/dataset/2024-04-11 Chemiluminescence.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:445b6da272aece5b854a76d1b99d48951805e5671e535a9752f187ee43aed06c +size 443308 diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..57ee2b06f05c0705b6951be1a4f0b5a18129ed65 Binary files /dev/null and b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx differ diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/protocols/.gitkeep b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_4_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md new file mode 100644 index 0000000000000000000000000000000000000000..93dd9aa986be0e84d5a9d25857d8229643eccef4 --- /dev/null +++ b/assays/S4_4_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md @@ -0,0 +1,6 @@ +# Southern Blot + +* Southern blot was performed as described previously [Morales et al. 2023]. +* Each DNA sample contained 7 µg of purified DNA. The DNA was digested using restriction enzymes BclI and SpeI or only BamHI (New England Biolabs). +* A 393 bp DIG-labelled probe against the blasticidin resistance gene (blastR) was generated by PCR using primers 3174 and 3175 (see S3 Table) and the DIG DNA Labelling Mixture (Roche) according to the manufacturer’s instructions. +* Hybridization was conducted at a temperature of 48 °C. \ No newline at end of file diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/README.md b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/.gitkeep b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.pzf b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.pzf new file mode 100644 index 0000000000000000000000000000000000000000..480287ab7ad51ab1b1608e711c96c4df47a75b8b --- /dev/null +++ b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.pzf @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:5f30f7b780c1cf8b30107a39439f61749503ca79993bfba1f9e75d92e3b48735 +size 157067 diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.xlsx b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..84a833555fd911e9d30ff15120fae5756c294072 Binary files /dev/null and b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/dataset/Growth curve_WT-Adea319-Adea400.xlsx differ diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/isa.assay.xlsx b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..0071ffbb3505e49ea0ceca0004ce851ab08b4822 Binary files /dev/null and b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/isa.assay.xlsx differ diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/protocols/.gitkeep b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/protocols/Protocol_Growth curve.md b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/protocols/Protocol_Growth curve.md new file mode 100644 index 0000000000000000000000000000000000000000..e0ae8689cd88ed3c5363ee2bbf273cb4dd203c1b --- /dev/null +++ b/assays/S4_5_Growth-curves-of-WT-Adea319-and-Adea400/protocols/Protocol_Growth curve.md @@ -0,0 +1,12 @@ +# Growth curves + +* For the analysis of the growth behavior of different A. deanei strains, precultures were grown to mid-exponential phase (approximately 1-5 x 10^7 cells/ml) with all selective drugs that would allow growth of the specific strains. +* Then, three replicates were prepared with 5 ml BHI plus hemin and antibiotics as indicated below and inoculated to an initial cell density of 1 x 10^5 cells/ml. +* The tubes were incubated at 28 °C without agitation and the cell density was measured every 20-24 h using the MultiSizer 4e (Beckman Coulter). + +## Antibiotic concentrations (final concentrations): + +* Neomycin (G418, Sigma-Aldrich): 500 µg/ml +* Hygromycin B (Invivogen): 500 µg/ml +* Blasticidin (Invivogen): 75 µg/ml +* Nourseothricin (Carl Roth): 200 µg/ml \ No newline at end of file diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/README.md b/assays/S4_6_Knock-out-strategy-for-T7RNAP/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/dataset/.gitkeep b/assays/S4_6_Knock-out-strategy-for-T7RNAP/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/dataset/2024-03-01 SYBR Safe stained agarose gel.tif b/assays/S4_6_Knock-out-strategy-for-T7RNAP/dataset/2024-03-01 SYBR Safe stained agarose gel.tif new file mode 100644 index 0000000000000000000000000000000000000000..50f4ae572da07d464124ebb9046e5365a1deed44 --- /dev/null +++ b/assays/S4_6_Knock-out-strategy-for-T7RNAP/dataset/2024-03-01 SYBR Safe stained agarose gel.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:88224f572a99e850361a32de8220657ab22d7dccfb1c21efd627296eb522fa14 +size 385684 diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/isa.assay.xlsx b/assays/S4_6_Knock-out-strategy-for-T7RNAP/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..edabd5ce86c789707aa11244978dc73180a25b0c Binary files /dev/null and b/assays/S4_6_Knock-out-strategy-for-T7RNAP/isa.assay.xlsx differ diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/protocols/.gitkeep b/assays/S4_6_Knock-out-strategy-for-T7RNAP/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_6_Knock-out-strategy-for-T7RNAP/protocols/Protocol_Cloning.md b/assays/S4_6_Knock-out-strategy-for-T7RNAP/protocols/Protocol_Cloning.md new file mode 100644 index 0000000000000000000000000000000000000000..6d37c3acb20497d30c723910c39a82d5b3b6cd15 --- /dev/null +++ b/assays/S4_6_Knock-out-strategy-for-T7RNAP/protocols/Protocol_Cloning.md @@ -0,0 +1,11 @@ +# Generation of plasmids (cloning) + +* All plasmids for this study were products of Gibson [Gibson et al. 2009] or Golden Gate [Engler et al. 2008] assemblies as described previously [Morales et al. 2023]. +* The cloning fragments, which were needed for the assemblies, were generated by PCR using the Phusion polymerase (New England Biolabs). Primers and templates are listed in S2 Table. +* The correct sizes of cloning fragments were verified by electrophoresis. +* PCR products matching the expected sizes were excised from the gel and purified using the Monarch DNA Gel Extraction kit (New England Biolabs). The concentration of the DNA was determined using the NanoPhotometer NP80 (Implen). +* The molar concentration of the insert(s) usually exceeded the vector backbone concentration by a factor of 3. +* After the assembly, 1 µl of DpnI was added to each assembly mix to remove residual PCR template present in the mix. +* Then, E. coli TOP10 cells were transformed with the assembly mix and recombinant cells selected on lysogeny broth (LB) plates containing 100 µg/ml ampicillin. +* Recovered clones were inoculated in 5 ml LB liquid medium with ampicillin (see above) and used for plasmid purification (Monarch Plasmid Miniprep kit, New England Biolabs). +* Plasmids were verified by control digests and sequencing (Microsynth). \ No newline at end of file diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/README.md b/assays/S4_7_Verification-of-knock-out-by-PCR/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/dataset/.gitkeep b/assays/S4_7_Verification-of-knock-out-by-PCR/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/dataset/2024-03-01 SYBR Safe stained agarose gel.tif b/assays/S4_7_Verification-of-knock-out-by-PCR/dataset/2024-03-01 SYBR Safe stained agarose gel.tif new file mode 100644 index 0000000000000000000000000000000000000000..50f4ae572da07d464124ebb9046e5365a1deed44 --- /dev/null +++ b/assays/S4_7_Verification-of-knock-out-by-PCR/dataset/2024-03-01 SYBR Safe stained agarose gel.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:88224f572a99e850361a32de8220657ab22d7dccfb1c21efd627296eb522fa14 +size 385684 diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/isa.assay.xlsx b/assays/S4_7_Verification-of-knock-out-by-PCR/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..a3f6f63131051e309284b23902855b48c57d180f Binary files /dev/null and b/assays/S4_7_Verification-of-knock-out-by-PCR/isa.assay.xlsx differ diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/protocols/.gitkeep b/assays/S4_7_Verification-of-knock-out-by-PCR/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_7_Verification-of-knock-out-by-PCR/protocols/Protocol_PCR.md b/assays/S4_7_Verification-of-knock-out-by-PCR/protocols/Protocol_PCR.md new file mode 100644 index 0000000000000000000000000000000000000000..df21ea168213193a8a1c160cad1f9fc88223ad16 --- /dev/null +++ b/assays/S4_7_Verification-of-knock-out-by-PCR/protocols/Protocol_PCR.md @@ -0,0 +1,9 @@ +# Polymerase chain reaction + +* All PCR reactions contained 100 ng for plasmid DNA or 50 ng of genomic DNA. +* For the amplification the Phusion polymerase (New England Biolabs) was used according to the manufacturer's instructions. +* The primer concentrations equaled 0.5 µM per primer. +* All reactions contained 1.25 M betaine as PCR enhancer. +* PCR steps: Denaturation at 95 °C for 20 s per cycle, Annealing at primer specific temperature for 20 s per cycle, elongation at 72 °C for 30 s per 1 kb +* Number of cycles: 30 +* TD-PCR for verification of genomic insertion (in delta-amastin locus): 72-68 °C annealing temperature for 2 cycles each, followed by 25 cycles at 60 °C, elongation time: 4 min \ No newline at end of file diff --git a/assays/S4_8_Growth-curves-of-WT-Adea373-and-Adea442/README.md b/assays/S4_8_Growth-curves-of-WT-Adea373-and-Adea442/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S4_8_Growth-curves-of-WT-Adea373-and-Adea442/dataset/.gitkeep b/assays/S4_8_Growth-curves-of-WT-Adea373-and-Adea442/dataset/.gitkeep new file mode 100644 index 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0000000000000000000000000000000000000000..e0ae8689cd88ed3c5363ee2bbf273cb4dd203c1b --- /dev/null +++ b/assays/S4_8_Growth-curves-of-WT-Adea373-and-Adea442/protocols/Protocol_Growth curve.md @@ -0,0 +1,12 @@ +# Growth curves + +* For the analysis of the growth behavior of different A. deanei strains, precultures were grown to mid-exponential phase (approximately 1-5 x 10^7 cells/ml) with all selective drugs that would allow growth of the specific strains. +* Then, three replicates were prepared with 5 ml BHI plus hemin and antibiotics as indicated below and inoculated to an initial cell density of 1 x 10^5 cells/ml. +* The tubes were incubated at 28 °C without agitation and the cell density was measured every 20-24 h using the MultiSizer 4e (Beckman Coulter). + +## Antibiotic concentrations (final concentrations): + +* Neomycin (G418, Sigma-Aldrich): 500 µg/ml +* Hygromycin B (Invivogen): 500 µg/ml +* Blasticidin (Invivogen): 75 µg/ml +* Nourseothricin (Carl Roth): 200 µg/ml \ No newline at end of file diff --git a/assays/S5_1_Genomic-map-of-Adea456/README.md b/assays/S5_1_Genomic-map-of-Adea456/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_1_Genomic-map-of-Adea456/dataset/.gitkeep b/assays/S5_1_Genomic-map-of-Adea456/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_1_Genomic-map-of-Adea456/isa.assay.xlsx b/assays/S5_1_Genomic-map-of-Adea456/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..e7c66e7e04a9a7c498299f47a7e2860591ac4da5 Binary files /dev/null and b/assays/S5_1_Genomic-map-of-Adea456/isa.assay.xlsx differ diff --git a/assays/S5_1_Genomic-map-of-Adea456/protocols/.gitkeep b/assays/S5_1_Genomic-map-of-Adea456/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_1_Genomic-map-of-Adea456/protocols/Protocol_Cloning.md b/assays/S5_1_Genomic-map-of-Adea456/protocols/Protocol_Cloning.md new file mode 100644 index 0000000000000000000000000000000000000000..6d37c3acb20497d30c723910c39a82d5b3b6cd15 --- /dev/null +++ b/assays/S5_1_Genomic-map-of-Adea456/protocols/Protocol_Cloning.md @@ -0,0 +1,11 @@ +# Generation of plasmids (cloning) + +* All plasmids for this study were products of Gibson [Gibson et al. 2009] or Golden Gate [Engler et al. 2008] assemblies as described previously [Morales et al. 2023]. +* The cloning fragments, which were needed for the assemblies, were generated by PCR using the Phusion polymerase (New England Biolabs). Primers and templates are listed in S2 Table. +* The correct sizes of cloning fragments were verified by electrophoresis. +* PCR products matching the expected sizes were excised from the gel and purified using the Monarch DNA Gel Extraction kit (New England Biolabs). The concentration of the DNA was determined using the NanoPhotometer NP80 (Implen). +* The molar concentration of the insert(s) usually exceeded the vector backbone concentration by a factor of 3. +* After the assembly, 1 µl of DpnI was added to each assembly mix to remove residual PCR template present in the mix. +* Then, E. coli TOP10 cells were transformed with the assembly mix and recombinant cells selected on lysogeny broth (LB) plates containing 100 µg/ml ampicillin. +* Recovered clones were inoculated in 5 ml LB liquid medium with ampicillin (see above) and used for plasmid purification (Monarch Plasmid Miniprep kit, New England Biolabs). +* Plasmids were verified by control digests and sequencing (Microsynth). \ No newline at end of file diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/README.md b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/.gitkeep b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-09 SYBR Safe stained gel.tif b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-09 SYBR Safe stained gel.tif new file mode 100644 index 0000000000000000000000000000000000000000..dfc6e5d65e9d40ea5e742329458b39653986fd2c --- /dev/null +++ b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-09 SYBR Safe stained gel.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:63a7d5f268e2e729dde12c54d5622da990ae1b7d42dce11d68510326f61e2ff2 +size 487058 diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-13 Chemiluminscence.tif b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-13 Chemiluminscence.tif new file mode 100644 index 0000000000000000000000000000000000000000..ccf335a3efdb8c45155a931417a82bf9e588ab5a --- /dev/null +++ b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/dataset/2025-01-13 Chemiluminscence.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1996af08947ca208b33ee3d9b9093ad785371bbe3a12cca83b876434e4aa5c76 +size 203890 diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..aaa6bf7ed9928cd4e2d16299487e546798fe366d Binary files /dev/null and b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/isa.assay.xlsx differ diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/protocols/.gitkeep b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_2_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md new file mode 100644 index 0000000000000000000000000000000000000000..93dd9aa986be0e84d5a9d25857d8229643eccef4 --- /dev/null +++ b/assays/S5_2_Verification-of-insertion-by-Southern-Blot/protocols/Protocol_Southern Blot.md @@ -0,0 +1,6 @@ +# Southern Blot + +* Southern blot was performed as described previously [Morales et al. 2023]. +* Each DNA sample contained 7 µg of purified DNA. The DNA was digested using restriction enzymes BclI and SpeI or only BamHI (New England Biolabs). +* A 393 bp DIG-labelled probe against the blasticidin resistance gene (blastR) was generated by PCR using primers 3174 and 3175 (see S3 Table) and the DIG DNA Labelling Mixture (Roche) according to the manufacturer’s instructions. +* Hybridization was conducted at a temperature of 48 °C. \ No newline at end of file diff --git a/assays/S5_3_Growth-curves-of-WT-and-Adea456/README.md b/assays/S5_3_Growth-curves-of-WT-and-Adea456/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_3_Growth-curves-of-WT-and-Adea456/dataset/.gitkeep b/assays/S5_3_Growth-curves-of-WT-and-Adea456/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_3_Growth-curves-of-WT-and-Adea456/dataset/Growth curve_WT-Adea456.pzf 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10^7 cells/ml) with all selective drugs that would allow growth of the specific strains. +* Then, three replicates were prepared with 5 ml BHI plus hemin and antibiotics as indicated below and inoculated to an initial cell density of 1 x 10^5 cells/ml. +* The tubes were incubated at 28 °C without agitation and the cell density was measured every 20-24 h using the MultiSizer 4e (Beckman Coulter). + +## Antibiotic concentrations (final concentrations): + +* Neomycin (G418, Sigma-Aldrich): 500 µg/ml +* Hygromycin B (Invivogen): 500 µg/ml +* Blasticidin (Invivogen): 75 µg/ml +* Nourseothricin (Carl Roth): 200 µg/ml \ No newline at end of file diff --git a/assays/S5_4_Epifluorescence-microscopy-of-Adea126-and-Adea456/README.md b/assays/S5_4_Epifluorescence-microscopy-of-Adea126-and-Adea456/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git 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0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_4_Epifluorescence-microscopy-of-Adea126-and-Adea456/protocols/Protocol_Epifluorescence microscopy.md b/assays/S5_4_Epifluorescence-microscopy-of-Adea126-and-Adea456/protocols/Protocol_Epifluorescence microscopy.md new file mode 100644 index 0000000000000000000000000000000000000000..a649946509b355c27e4854bed943428b7ae56204 --- /dev/null +++ b/assays/S5_4_Epifluorescence-microscopy-of-Adea126-and-Adea456/protocols/Protocol_Epifluorescence microscopy.md @@ -0,0 +1,6 @@ +# Epifluorescence microscopy + +* Microscopy slides were prepared from mid-exponential cultures (approximately 1-5 x 10^7 cells/ml) as described before [Morales et al. 2023]. +* Images were taken with the Axio Imager A.2 equipped with the AxioCam MRm and an Illuminator HXP 120 V (all instruments from Zeiss) and processed with Zen Blue v2.5 (Zeiss) as described previously [Morales et al. 2023]. +* A. deanei WT cells served as negative control and its autofluorescence was used to define a suitable fluorescence threshold. +* For post-processing of the pictures Fiji [Schindelin et al. 2012] was used. \ No newline at end of file diff --git a/assays/S5_5_Quantification-of-fluorescent-cells/README.md b/assays/S5_5_Quantification-of-fluorescent-cells/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_5_Quantification-of-fluorescent-cells/dataset/.gitkeep b/assays/S5_5_Quantification-of-fluorescent-cells/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_5_Quantification-of-fluorescent-cells/dataset/Quantification_mSc strains.xlsx b/assays/S5_5_Quantification-of-fluorescent-cells/dataset/Quantification_mSc strains.xlsx new file mode 100644 index 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a/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/dataset/2025-01-17 Stainfree gel image.tif b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/dataset/2025-01-17 Stainfree gel image.tif new file mode 100644 index 0000000000000000000000000000000000000000..2985163a7aab873b9f383d292af2ab3ef711ff92 --- /dev/null +++ b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/dataset/2025-01-17 Stainfree gel image.tif @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:73a6f1d4a08f54ee49e0d075ad943bf7d969b02253229de1d720373cca04c391 +size 316102 diff --git a/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/isa.assay.xlsx b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..d70e2bd47ea5dad7a4f8516846a5f6f26a099603 Binary files /dev/null and b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/isa.assay.xlsx differ diff --git a/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/protocols/.gitkeep b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/protocols/Protocol_In gel fluorescence assay.md b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/protocols/Protocol_In gel fluorescence assay.md new file mode 100644 index 0000000000000000000000000000000000000000..4985608f46eba7cd9e9005cc2f201db315501090 --- /dev/null +++ b/assays/S5_6_In-gel-fluorescence-assay-of-Adea126-and-Adea456/protocols/Protocol_In gel fluorescence assay.md @@ -0,0 +1,11 @@ +# In gel fluorescence assay + +* For the detection of in-gel fluorescence of mScarlet, samples were prepared as described by Sanial et al. 2024 with minor modifications. +* First, 15 ml cultures of the relevant strains were grown to mid-exponential phase, cells were harvested by centrifugation, washed twice using 1x PBS, and resuspended in 1 ml 1x PBS. +* Before cell lysis, cOmplete EDTA-free protease inhibitor tabs (Roche) and a few grains of DNase (Carl Roth) were added and the cells were lysed by sonication using the UP100H (Hielscher) with 5 x 18 pulses (cycle 0.8, amplitude ~60 %, MS1 sonotrode). +* Cell debris and intact cells were removed by centrifugation (3000 rpm, 4 °C, 5 min) and the protein concentration of the supernatant was determined using the Pierce 660nm Protein Assay Reagent (ThermoFisher) and the FLUOstar Omega Plate Reader (BMG Labtech). +* 25 µg of protein were mixed with Lämmli sample buffer (final concentrations: 50 mM Tris-HCl, pH 6.8, 10% v/v glycerol, 2% SDS w/v, 2.5 mM EDTA, and 0.005% w/v bromophenol blue, plus 12.5 mM dithiothreitol), heated to 50 °C for 5 min and applied to a 12.5 % SDS polyacrylamid gel containing 0.5 % v/v TCE. +* The electrophoresis ran at 40 mA until the marker bands (Prestained Ladder, ThermoFisher) were fully resolved. +* Finally, mScarlet fluorescence was recorded with the ChemiDoc MP (Biorad) using the UV tray and the “Cy3†filter. +* Afterwards, loading of the gel was visualized using TCE fluorescence with the UV tray and the “Stain-free gel†setting of the ChemiDoc MP (Biorad). +* The intensities of the mScarlet and TCE bands were estimated with Fiji [Schindelin et al. 2012] and normalized by the TCE signals. \ No newline at end of file diff --git a/assays/S5_7_Quantification-of-in-gel-assay/README.md b/assays/S5_7_Quantification-of-in-gel-assay/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/S5_7_Quantification-of-in-gel-assay/dataset/.gitkeep b/assays/S5_7_Quantification-of-in-gel-assay/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git 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