ActinoBase - User contributions [en-gb]

PCRISPR-dCas9

2021-05-18T16:26:03Z

H Otani: Created page with "==References== #Tong Y, Charusanti P, Zhang L, Weber T, Lee SY. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes. ''ACS Synth Biol'' 2015 Sep 18;4(9):1020-9. doi: 10.102..."

==References==
#Tong Y, Charusanti P, Zhang L, Weber T, Lee SY. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes. ''ACS Synth Biol'' 2015 Sep 18;4(9):1020-9. doi: 10.1021/acssynbio.5b00038. Epub 2015 Apr 7. 10.1021/acssynbio.5b00038

PCRISPR-Cas9

2021-05-18T16:25:53Z

PTYM18

2021-05-18T16:23:06Z

H Otani: Created page with "==References== #Onaka, H., Taniguchi, S., Ikeda, H., Igarashi, Y., and Furumai, T. (2003). pTOYAMAcos, pTYM18, and pTYM19, actinomycete-''Escherichia coli'' integrating vector..."

==References==
#Onaka, H., Taniguchi, S., Ikeda, H., Igarashi, Y., and Furumai, T. (2003). pTOYAMAcos, pTYM18, and pTYM19, actinomycete-''Escherichia coli'' integrating vectors for heterologous gene expression. ''J Antibiot (Tokyo)'', 56, 950-956 DOI: 10.7164/antibiotics.56.950

PTYM19

2021-05-18T16:22:52Z

PTOYAMAcos

2021-05-18T16:22:30Z

Plasmids

2021-05-18T16:18:37Z

H Otani: /* Plasmids used in Streptomyces */

[[File:Actinobase-icons-trans_Plasmids.png|150px|Plasmids|link=Plasmids]]

Various plasmids have been developed for use in Streptomyces (most will also contain an origin of replication that functions in Escherichia coli, for cloning, general maintenance, and [[intergeneric conjugation into Streptomyces]]). Many of the plasmids commonly used for Streptomyces work are '''integrative''': they insert into a specific site on the Streptomyces chromosome using a [[site-specific phage recombinase]].

The following is an alphabetical list of a number of useful vectors - see also [[Overview of Streptomyces vectors]].

==Plasmids used in Streptomyces==

[[pCAP03]]

[[pCRISPomyces-2]]

[[pCRISPR-Cas9]]

[[pCRISPR-dCas9]]

[[pESAC13]]

[[pIJ101]]

[[pIJ8660]]

[[pIJ10257]]

[[pIJ12738]]

[[pIJ12742]]

[[pIJ6902]]

[[pKC1132]]

[[pMF96]]

[[pMS82]]

[[pRT802]]

[[pSET152]]

[[pSS170]]

[[pTOYAMAcos]]

[[pTYM18]]

[[pTYM19]]

[[Supercos1]]

[[pGM9]]

[[pGM190]]

==Plasmids used for PCR targeting (Redirect)==

[http://streptomyces.org.uk/redirect/index.html Redirect protocol and template information]

[[pIJ773]]

==Plasmids used for heterologous protein expression in E. coli==

The following plasmids are frequently used to express proteins heterologously in E. coli, e.g. for two-hybrid analysis or protein purification.

[[pET28a]]

Vectors for Bacterial two-hybrid experiments
[[pKT25/pKNT25]] | [[pUT18/pUT18C]]

Recruitment

2020-07-15T19:13:51Z

H Otani: /* Recruitment */

=Recruitment=
Please feel free to post information and links to job opportunities etc below:
*Please also remember to remove the links once filled or application expires.

Postgraduate opportunities at the John Innes Centre: find out more [https://www.jic.ac.uk/training-careers/postgraduate-opportunities/current-postgraduate-research-opportunities/ here]

=Opportunities=
Applied molecular microbiology summer school: 'Microbial Specialised Metabolites: Discovery, Biosynthesis and Evolution'
*This is a fantastic summer school for early career researchers studying natural products, which takes place in Dubrovnik 5-12 September 2020. Applications close 14 February 2020.
*Find out more and apply [https://www.jic.ac.uk/training-careers/summer-schools/applied-molecular-microbiology/ here]

If you wish to advertise an opportunity, but do not have editor access to ActinoBase, please see the [[Admin contact page]] to either request an editors account, or request your oppotunity is advertised here.

Protoplast Transformation

2020-03-31T05:26:48Z

H Otani: /* Instructions */

==Materials==

*[[P buffer]]
*1 g polyethylene glycol 1000 (PEG)
*[[R5]] or [[R2YE]] plate

==Instructions==

#Follow the instruction in [[Protoplasts Formation, Regeneration and Transformation]]
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 28-30ºC overnight (typically 12-18 hours).
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

Teaching and learning

2020-03-25T17:15:50Z

H Otani: /* Podcasts */

=Teaching and Learning=

This is the hub for #ActinoBase teaching and learning resources: educational content, outreach, and more! We welcome contributions from the community, so please [[Admin contact page|contact]] us with any suggestions or if you would like to contribute content to these pages.

==Practical protocols (undergrad labs)==

==Undergraduate student projects==

*[https://seaphages.org/ SEA-PHAGES], an HHMI undergraduate course featuring the discovery and isolation of actinobacteriophages
*[https://www.strath.ac.uk/viprojects/drugdiscovery/ the Strathclyde VIP Drug Discovery Project], using hyperspectral imaging to screen for novel antibiotic producers

==Spotlight: teaching antibiotics & AMR==

Are you looking for videos to use in your courses, inspiration for classroom or workshop activities, or other resources to help you with your teaching? Please check out our collection of [[Teaching and Learning Resources: Antibiotics and AMR]].

==Other teaching and learning resources==
*[https://microbiologyonline.org/teachers Microbiology Society resources for teachers]
*[https://microbiologyonline.org/students Microbiology Society resources for students]
*[https://www.asmscience.org/VisualLibrary the American Society for Microbiology's resource library]
*[https://www.asmscience.org/content/journal/jmbe Journal of Microbiology and Biology Education]
*[https://schaechter.asmblog.org/schaechter/teachers-corner-1/ Small Things Considered: Teacher's Corner]

=Outreach=
[https://www.uea.ac.uk/research/explore-uea-research/antibiotic-hunters Antibiotic Hunters]: Huge congratulations to Prof. Matt Hutchings for winning the [https://microbiologysociety.org/news/society-news/professor-matt-hutchings-wins-the-2019-microbiology-outreach-prize.html 2019 Outreach Prize]!

==Outreach Events Calendar==

Are you presenting your actinobacteria research at a science festival, [https://pintofscience.co.uk/ Pint of Science], [https://twitter.com/soapboxscience?lang=en Soapbox Science], or other outreach event? Let us know so that we can add you to our [[Outreach Events Calendar]]!

=Student pages=
If you would like to use ActinoBase for a wiki creation/editing assignment as part of a course or workshop, please [[Admin contact page|contact]] us.

=Other resources=

==Blogs==
*[https://microbiologysociety.org/news-press/microbe-post.html Microbe Post]
*[https://schaechter.asmblog.org/ Small Things Considered]

==Podcasts==
*[https://soundcloud.com/microbiosoc Microbe Talk]
*[https://www.asm.org/Podcasts/TWiM This Week in Microbiology]
*[https://www.asm.org/Podcasts/BacterioFiles BacterioFiles]
*[https://www.asm.org/Podcasts/microTalk microTalk]
*[https://www.asm.org/Podcasts/MTM Meet the Microbiologist]
*[http://naturalprodcast.com/ Natural Prodcast]

==Microbial Art==
The range of colored pigments produced by different actinobacteria, coupled with their tendency for non-lateral growth, make them suitable for creating biological art. Creating and observing biological art can be used as a vehicle to engage people with diverse backgrounds and interests in both concepts and techniques related to chemistry and biology.[https://doi.org/10.1371/journal.pbio.1000510 1]

“Agar art” has been shown to be an effective method for increasing confidence in students being introduced to microbiology.[https://doi.org/10.1093/femsle/fnx245 2] Similarly, as long as proper sterile conditions are available, creating art using ''Streptomyces'' has been an engaging activity for introducing high school students to microbiology and working with elementary school populations. BioArt employing actinobacteria can therefore be used to introduce both younger students and artists to science, including those from traditionally marginalized identities in STEM.[https://doi.org/10.1371/journal.pbio.3000061 3]

The following strains are streaked, using sterile technique, on R5 media in order to “draw” with their pigments. The strains can also be streaked on a modified, less expensive media using food-grade agar and/or chicken broth. Pigments appear after 3-7 days of growth.[https://doi.org/10.1371/journal.pbio.1000510 1] All ''Streptomyces'' strains are classified as Biosafety Level 1 (BSL-1) organisms, and are of minimal hazard to humans.[https://doi.org/10.1002/9780471729259.mc10e01s18 4]
* ''Streptomyces coelicolor'' (Blue, Red under acidic conditions)
* ''Streptomyces violaceoruber'' (Purple, Pink under acidic conditions)
* ''Streptomyces roseofulvus'' (Yellow)

The colored pigments can also be extracted, dried, and resuspended in acrylic to produce bio-based paint.

Using standard 100 mm petri dishes allows for smaller-scale activities, which is often preferable when introducing proper bacterial streaking technique so that participants can create multiple pieces of art. However, using larger plates allows experienced artists to create much more detailed and intricate drawings.

[[File:BioArt.png|450px]][https://doi.org/10.1371/journal.pbio.3000061 3] [[File:BioArtElvis.PNG|310px]][https://doi.org/10.1371/journal.pbio.1000510 1]

The characteristic colored pigments produced by actinobacterial strains enable concurrent learning opportunities about molecular color and natural products. While leading or introducing the activity, participants may be interested to learn that biosynthesized molecules are responsible for the drawing’s vibrant color and that these molecules can have antibiotic and anticancer properties.

References:

1. Charkoudian, L.K., Fitzgerald, J.T., Khosla, C., Champlin, A., 2010. In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond. PLoS Biol 8, e1000510. https://doi.org/10.1371/journal.pbio.1000510

2. Adkins, S.J., Rock, R.K., Morris, J.J., 2018. Interdisciplinary STEM education reform: dishing out art in a microbiology laboratory. FEMS Microbiology Letters 365. https://doi.org/10.1093/femsle/fnx245

3. Lopes, L.E., Waldis, S.J., Terrell, S.M., Lindgren, K.A., Charkoudian, L.K., 2018. Vibrant symbiosis: Achieving reciprocal science outreach through biological art. PLoS Biol 16, e3000061. https://doi.org/10.1371/journal.pbio.3000061

4. Shepherd, M.D., Kharel, M.K., Bosserman, M.A., Rohr, J., 2010. Laboratory Maintenance of Streptomyces Species. Current Protocols in Microbiology 18. https://doi.org/10.1002/9780471729259.mc10e01s18

RNA Sequencing

2020-03-25T00:18:08Z

H Otani:

==Instructions==

This procedure is an example and the optimal library preparation and data analysis procedures can be different depending on the type of the DNA sequencer used and purpose of the experiment.

#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.
#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.
#Deplete rRNA using [https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html Illumina Ribo-Zero Plus rRNA Depletion Kit] or a similar product
#Create Illumina RNA-seq libraries using [https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html TruSeq RNA Library Prep Kit].
#Sequence the libraries using an Illumina system such as [https://www.illumina.com/systems/sequencing-platforms/novaseq.html NovaSeq 6000 Sequencing System].
#Trim low quality subsequences from reads using [http://hannonlab.cshl.edu/fastx_toolkit/ FASTX-Toolkit] (single-end) or [https://research.bioinformatics.udel.edu/genomics/ngsShoRT/ ngsShoRT] (single- or paired-end).
#Align reads with the reference genome using [http://daehwankimlab.github.io/hisat2/ HISAT2] or [http://bowtie-bio.sourceforge.net/bowtie2/index.shtml Bowtie2].
#Convert the SAM files into BAM files and sort and index the BAM files using [http://www.htslib.org/ Samtools].
#Count the number of reads overlapping with each feature such as CDS or tRNA gene using [http://subread.sourceforge.net/ featureCounts] or [https://htseq.readthedocs.io/en/master/ HTSeq].
#Perform a differential gene expression analysis using a Bioconductor package such as [https://bioconductor.org/packages/release/bioc/html/DESeq2.html DESeq2].

RNA Sequencing

2020-03-25T00:07:56Z

H Otani: Created page with "#Extract and purify RNA samples according to RNA extraction from Actinobacteria. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In..."

#Extract and purify RNA samples according to [[RNA extraction from Actinobacteria]]. Add [https://www.thermofisher.com/order/catalog/product/4456740#/4456740 ERCC RNA Spike-In Mix] if necessary.
#Check the quality of RNA samples using [https://www.chem.agilent.com/store/productDetail.jsp?catalogId=5067-1511&catId=SubCat2ECS_227716 Agilent RNA 6000 Nano Kit] or by running them on an agarose gel.
#Deplete rRNA using [https://www.illumina.com/products/by-type/accessory-products/ribo-zero-plus-rrna-depletion.html Illumina Ribo-Zero Plus rRNA Depletion Kit] or a similar product
#Create Illumina RNA-seq libraries using [https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html TruSeq RNA Library Prep Kit].
#Sequence the libraries using an Illumina system such as [https://www.illumina.com/systems/sequencing-platforms/novaseq.html NovaSeq 6000 Sequencing System].
#Trim low quality subsequences from reads using [http://hannonlab.cshl.edu/fastx_toolkit/ FASTX-Toolkit] (single-end) or [https://research.bioinformatics.udel.edu/genomics/ngsShoRT/ ngsShoRT] (single- or paired-end).
#Align reads with the reference genome using [http://daehwankimlab.github.io/hisat2/ HISAT2] or [http://bowtie-bio.sourceforge.net/bowtie2/index.shtml Bowtie2].
#Convert the SAM files into BAM files and sort and index the BAM files using [http://www.htslib.org/ Samtools].
#Count the number of reads overlapping with each feature such as CDS or tRNA gene using [http://subread.sourceforge.net/ featureCounts] or [https://htseq.readthedocs.io/en/master/ HTSeq].
#Perform a differential gene expression analysis using a Bioconductor package such as [https://bioconductor.org/packages/release/bioc/html/DESeq2.html DESeq2].

Natural Products

2020-03-24T23:43:03Z

H Otani: Created page with "Cloning, Transformation and Expression of Gene Clusters Metabolite extractions Production of microbial volatile organic compounds"

[[Cloning, Transformation and Expression of Gene Clusters]]

[[Metabolite extractions]]

[[Production of microbial volatile organic compounds]]

Protocols

2020-03-24T23:41:47Z

H Otani: /* Alphabetical list of protocols */

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

=Protocols organised by category=

==[[Biochemistry]]==

*[[GUS assay]] (β-Glucuronidase assay)

==Microbiology==

*[[Characterisation of Actinomycetes | General Characterisaton of Actinomycetes]]

*[[Exploration | ''Streptomyces'' Exploration]]

*[[General Growth and Maintenance of Streptomyces sp.]]

*[[Isolating Actinomycetes from the environment]]

For a comprehensive guide to actinomycete characterisation see the [https://www.dsmz.de/collection/catalogue/microorganisms/special-groups-of-organisms/compendium-of-actinobacteria Compendium of Actinobacteria] from [https://www.helmholtz-hzi.de/en/research/research-topics/anti-infectives/microbial-strain-collection/our-research/#anchorsection Prof. Joachim Wink] at the German Collection of Microorganisms & Cell Cultures ([https://www.dsmz.de/ DSMZ]). This resouce provides protocols for characterising a wide range of features for new isolates, but may also be applicable more broadly for example for phenotyping experiments. The tests include colony morphology, melanin production, salt tolerance, carbon utlilisation and much more. To download the guide please see the link below.

[https://actinobase.org/downloads/DSMZ.pdf DSMZ Compendium of Actinobacteria]

==[[Microscopy]]==

*[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

*[[Cultivating Actinobacteria for Microscopic Analysis]]

==[[Mutagenesis]]==

*[[CRISPR/Cas9-mediated genome editing]]

*[[Chemical mutagenesis]]

*[[Depletion strains]]

*[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

*[[Lambda-red mediated recombination using ssDNA]]

*[[Meganuclease I-SceI based system for gene deletions]]

*[[Protoplasts Formation, Regeneration and Transformation]]

*[["Quikchange" site-directed mutagenesis]]

*[[Random mutagenesis using a mutator strain]]

*[[Random mutagenesis using error-prone PCR]]

*[[Suicide vectors]]

*[[Transposons]]

*[[UV mutagenesis]]

==[[Natural Products]]==

*[[Cloning, Transformation and Expression of Gene Clusters]]

*[[Metabolite extractions]]

*[[Production of microbial volatile organic compounds]]

==[[Next Generation Sequencing]]==

*[[ChIP Sequencing]]
*[[ChIP-exo sequencing]]
https://www.nature.com/articles/s41467-018-05265-7/
*[[RNA Sequencing]]
*[[Differential RNA sequencing]]

==[[Nucleic Acid Methods]]==

*[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

*[[High GC PCR]]

*[[RNA extraction from Actinobacteria]]

*[[RNA interference]]

*[[Salting Out Genomic DNA Extraction Method]]

*[[Southern Blotting]]

==[[Protein Methods]]==

*[[Isolating extracellular protein from Streptomyces]]

=Alphabetical list of protocols=

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[Chemical mutagenesis]]

[[ChIP Seq]]

[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

[[Depletion strains]]

[[Exploration | ''Streptomyces'' Exploration]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[GUS assay]] (β-Glucuronidase assay)

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Isolating extracellular protein from Streptomyces]]

[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

[[Lambda-red mediated recombination using ssDNA]]

[[Meganuclease I-SceI based system for gene deletions]]

[[Metabolite extractions]]

[[Protoplasts Formation, Regeneration and Transformation]]

[["Quikchange" site-directed mutagenesis]]

[[Random mutagenesis using a mutator strain]]

[[Random mutagenesis using error-prone PCR]]

[[RNA extraction from Actinobacteria]]

[[Salting Out Genomic DNA Extraction Method]]

[[Southern Blotting]]

[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

[[Suicide vectors]]

[[Transposons]]

[[UV mutagenesis]]

Mutagenesis

2020-03-24T23:40:27Z

H Otani:

A number of different methods have been developed for random and directed mutagenesis in actinobacteria (most notably for Streptomyces coelicolor). Many involve an initial mutagenesis step performed in Escherichia coli, with subsequent transfer of the mutant allele(s) into the desired strain i.e. by [[protoplast transformation]] or [[intergeneric conjugation into Streptomyces]]. Others, such as CRISPR/Cas- or meganuclease I-SceI- mediated genome editing, can be performed directly in the actinobacterial strain of interest.

Your choice of mutagenesis method will depend largely on your intended use, and on whether you have an appropriate genetic screen/selection that you can use to identify your mutant(s) of interest.

[[CRISPR/Cas9-mediated genome editing]]

[[Chemical mutagenesis]]

[[Depletion strains]]

[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

[[Lambda-red mediated recombination using ssDNA]]

[[Meganuclease I-SceI based system for gene deletions]]

[[Protoplasts Formation, Regeneration and Transformation]]

[["Quikchange" site-directed mutagenesis]]

[[Random mutagenesis using a mutator strain]]

[[Random mutagenesis using error-prone PCR]]

[[Suicide vectors]]

[[Transposons]]

[[UV mutagenesis]]

Protocols

2020-03-24T23:38:40Z

H Otani: /* Mutagenesis */

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

=Protocols organised by category=

==[[Biochemistry]]==

*[[GUS assay]] (β-Glucuronidase assay)

==Microbiology==

*[[Characterisation of Actinomycetes | General Characterisaton of Actinomycetes]]

*[[Exploration | ''Streptomyces'' Exploration]]

*[[General Growth and Maintenance of Streptomyces sp.]]

*[[Isolating Actinomycetes from the environment]]

For a comprehensive guide to actinomycete characterisation see the [https://www.dsmz.de/collection/catalogue/microorganisms/special-groups-of-organisms/compendium-of-actinobacteria Compendium of Actinobacteria] from [https://www.helmholtz-hzi.de/en/research/research-topics/anti-infectives/microbial-strain-collection/our-research/#anchorsection Prof. Joachim Wink] at the German Collection of Microorganisms & Cell Cultures ([https://www.dsmz.de/ DSMZ]). This resouce provides protocols for characterising a wide range of features for new isolates, but may also be applicable more broadly for example for phenotyping experiments. The tests include colony morphology, melanin production, salt tolerance, carbon utlilisation and much more. To download the guide please see the link below.

[https://actinobase.org/downloads/DSMZ.pdf DSMZ Compendium of Actinobacteria]

==[[Microscopy]]==

*[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

*[[Cultivating Actinobacteria for Microscopic Analysis]]

==[[Mutagenesis]]==

*[[CRISPR/Cas9-mediated genome editing]]

*[[Chemical mutagenesis]]

*[[Depletion strains]]

*[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

*[[Lambda-red mediated recombination using ssDNA]]

*[[Meganuclease I-SceI based system for gene deletions]]

*[[Protoplasts Formation, Regeneration and Transformation]]

*[["Quikchange" site-directed mutagenesis]]

*[[Random mutagenesis using a mutator strain]]

*[[Random mutagenesis using error-prone PCR]]

*[[Suicide vectors]]

*[[Transposons]]

*[[UV mutagenesis]]

==[[Natural Products]]==

*[[Cloning, Transformation and Expression of Gene Clusters]]

*[[Metabolite extractions]]

*[[Production of microbial volatile organic compounds]]

==[[Next Generation Sequencing]]==

*[[ChIP Sequencing]]
*[[ChIP-exo sequencing]]
https://www.nature.com/articles/s41467-018-05265-7/
*[[RNA Sequencing]]
*[[Differential RNA sequencing]]

==[[Nucleic Acid Methods]]==

*[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

*[[High GC PCR]]

*[[RNA extraction from Actinobacteria]]

*[[RNA interference]]

*[[Salting Out Genomic DNA Extraction Method]]

*[[Southern Blotting]]

==[[Protein Methods]]==

*[[Isolating extracellular protein from Streptomyces]]

=Alphabetical list of protocols=

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[Chemical mutagenesis]]

[[ChIP Seq]]

[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

[[Depletion strains]]

[[Exploration | ''Streptomyces'' Exploration]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[GUS assay]] (β-Glucuronidase assay)

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Isolating extracellular protein from Streptomyces]]

[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

[[Lambda-red mediated recombination using ssDNA]]

[[Meganuclease I-SceI based system for gene deletions]]

[[Metabolite extractions]]

[["Quikchange" site-directed mutagenesis]]

[[Random mutagenesis using a mutator strain]]

[[Random mutagenesis using error-prone PCR]]

[[RNA extraction from Actinobacteria]]

[[Salting Out Genomic DNA Extraction Method]]

[[Southern Blotting]]

[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

[[Suicide vectors]]

[[Transposons]]

[[UV mutagenesis]]

Teaching and learning

2020-03-24T22:03:11Z

H Otani: /* Podcasts */

=Teaching and Learning=

This is the hub for #ActinoBase teaching and learning resources: educational content, outreach, and more! We welcome contributions from the community, so please [[Admin contact page|contact]] us with any suggestions or if you would like to contribute content to these pages.

==Practical protocols (undergrad labs)==

==Undergraduate student projects==

*[https://seaphages.org/ SEA-PHAGES], an HHMI undergraduate course featuring the discovery and isolation of actinobacteriophages
*[https://www.strath.ac.uk/viprojects/drugdiscovery/ the Strathclyde VIP Drug Discovery Project], using hyperspectral imaging to screen for novel antibiotic producers

==Spotlight: teaching antibiotics & AMR==

Are you looking for videos to use in your courses, inspiration for classroom or workshop activities, or other resources to help you with your teaching? Please check out our collection of [[Teaching and Learning Resources: Antibiotics and AMR]].

==Other teaching and learning resources==
*[https://microbiologyonline.org/teachers Microbiology Society resources for teachers]
*[https://microbiologyonline.org/students Microbiology Society resources for students]
*[https://www.asmscience.org/VisualLibrary the American Society for Microbiology's resource library]
*[https://www.asmscience.org/content/journal/jmbe Journal of Microbiology and Biology Education]
*[https://schaechter.asmblog.org/schaechter/teachers-corner-1/ Small Things Considered: Teacher's Corner]

=Outreach=
[https://www.uea.ac.uk/research/explore-uea-research/antibiotic-hunters Antibiotic Hunters]: Huge congratulations to Prof. Matt Hutchings for winning the [https://microbiologysociety.org/news/society-news/professor-matt-hutchings-wins-the-2019-microbiology-outreach-prize.html 2019 Outreach Prize]!

==Outreach Events Calendar==

Are you presenting your actinobacteria research at a science festival, [https://pintofscience.co.uk/ Pint of Science], [https://twitter.com/soapboxscience?lang=en Soapbox Science], or other outreach event? Let us know so that we can add you to our [[Outreach Events Calendar]]!

=Student pages=
If you would like to use ActinoBase for a wiki creation/editing assignment as part of a course or workshop, please [[Admin contact page|contact]] us.

=Other resources=

==Blogs==
*[https://microbiologysociety.org/news-press/microbe-post.html Microbe Post]
*[https://schaechter.asmblog.org/ Small Things Considered]

==Podcasts==
*[https://soundcloud.com/microbiosoc Microbe Talk]
*[https://www.asm.org/Podcasts/TWiM This Week in Microbiology]
*[https://www.asm.org/Podcasts/BacterioFiles BacterioFiles]
*[https://www.asm.org/Podcasts/microTalk microTalk]
*[https://www.asm.org/Podcasts/MTM Meet the Microbiologist]
*[https://open.spotify.com/show/3HzwKCSeyZM1WFlCmh70bA Natural Prodcast]

==Microbial Art==
The range of colored pigments produced by different actinobacteria, coupled with their tendency for non-lateral growth, make them suitable for creating biological art. Creating and observing biological art can be used as a vehicle to engage people with diverse backgrounds and interests in both concepts and techniques related to chemistry and biology.[https://doi.org/10.1371/journal.pbio.1000510 1]

“Agar art” has been shown to be an effective method for increasing confidence in students being introduced to microbiology.[https://doi.org/10.1093/femsle/fnx245 2] Similarly, as long as proper sterile conditions are available, creating art using ''Streptomyces'' has been an engaging activity for introducing high school students to microbiology and working with elementary school populations. BioArt employing actinobacteria can therefore be used to introduce both younger students and artists to science, including those from traditionally marginalized identities in STEM.[https://doi.org/10.1371/journal.pbio.3000061 3]

The following strains are streaked, using sterile technique, on R5 media in order to “draw” with their pigments. The strains can also be streaked on a modified, less expensive media using food-grade agar and/or chicken broth. Pigments appear after 3-7 days of growth.[https://doi.org/10.1371/journal.pbio.1000510 1] All ''Streptomyces'' strains are classified as Biosafety Level 1 (BSL-1) organisms, and are of minimal hazard to humans.[https://doi.org/10.1002/9780471729259.mc10e01s18 4]
* ''Streptomyces coelicolor'' (Blue, Red under acidic conditions)
* ''Streptomyces violaceoruber'' (Purple, Pink under acidic conditions)
* ''Streptomyces roseofulvus'' (Yellow)

The colored pigments can also be extracted, dried, and resuspended in acrylic to produce bio-based paint.

Using standard 100 mm petri dishes allows for smaller-scale activities, which is often preferable when introducing proper bacterial streaking technique so that participants can create multiple pieces of art. However, using larger plates allows experienced artists to create much more detailed and intricate drawings.

[[File:BioArt.png|450px]][https://doi.org/10.1371/journal.pbio.3000061 3] [[File:BioArtElvis.PNG|310px]][https://doi.org/10.1371/journal.pbio.1000510 1]

The characteristic colored pigments produced by actinobacterial strains enable concurrent learning opportunities about molecular color and natural products. While leading or introducing the activity, participants may be interested to learn that biosynthesized molecules are responsible for the drawing’s vibrant color and that these molecules can have antibiotic and anticancer properties.

References:

1. Charkoudian, L.K., Fitzgerald, J.T., Khosla, C., Champlin, A., 2010. In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond. PLoS Biol 8, e1000510. https://doi.org/10.1371/journal.pbio.1000510

2. Adkins, S.J., Rock, R.K., Morris, J.J., 2018. Interdisciplinary STEM education reform: dishing out art in a microbiology laboratory. FEMS Microbiology Letters 365. https://doi.org/10.1093/femsle/fnx245

3. Lopes, L.E., Waldis, S.J., Terrell, S.M., Lindgren, K.A., Charkoudian, L.K., 2018. Vibrant symbiosis: Achieving reciprocal science outreach through biological art. PLoS Biol 16, e3000061. https://doi.org/10.1371/journal.pbio.3000061

4. Shepherd, M.D., Kharel, M.K., Bosserman, M.A., Rohr, J., 2010. Laboratory Maintenance of Streptomyces Species. Current Protocols in Microbiology 18. https://doi.org/10.1002/9780471729259.mc10e01s18

Protoplast Transformation

2020-03-24T21:58:16Z

H Otani: /* Materials */

==Materials==

*[[P buffer]]
*1 g polyethylene glycol 1000 (PEG)
*[[R5]] or [[R2YE]] plate

==Instructions==

#Follow the instruction in [[Protoplasts Formation, Regeneration and Transformation]]
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 298-30ºC overnight.
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

Protoplast Transformation

2020-03-24T21:57:35Z

H Otani:

==Materials==

*[[P buffer]]
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
*[[R5]] or [[R2YE]] plate

==Instructions==

#Follow the instruction in [[Protoplasts Formation, Regeneration and Transformation]]
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 298-30ºC overnight.
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

Protoplasts Formation, Regeneration and Transformation

2020-03-24T21:55:58Z

H Otani: /* Materials */

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Protoplast Transformation

2020-03-24T21:55:21Z

H Otani: Created page with "#Follow the instruction in Protoplasts Formation, Regeneration and Transformation #Add 3.85 ml P buffer to 1 g PEG sterilised. #Add 200 µl PEG-P buffer mixture and a DNA..."

#Follow the instruction in [[Protoplasts Formation, Regeneration and Transformation]]
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 298-30ºC overnight.
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

Protoplasts Formation, Regeneration and Transformation

2020-03-24T21:53:41Z

H Otani: /* Instructions */

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool
*[[R5]] or [[R2YE]] plate

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Protoplasts Formation, Regeneration and Transformation

2020-03-24T21:52:54Z

H Otani: /* Materials */

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool
*[[R5]] or [[R2YE]] plate

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 298-30ºC overnight.
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Protoplasts Formation, Regeneration and Transformation

2020-03-24T21:52:04Z

H Otani: /* Instructions */

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].
#Add 3.85 ml P buffer to 1 g PEG sterilised.
#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
#Plate it onto [[R5]] or [[R2YE]].
#Incubate the plate at 298-30ºC overnight.
#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Protoplasts Formation, Regeneration and Transformation

2020-03-24T21:44:35Z

H Otani: /* Materials */

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

2019-08-30T21:22:00Z

H Otani:

Saccharopolyspora spinosa

2019-08-30T20:48:50Z

H Otani: Created page with "==Organism Page== === ''Saccharopolyspora spinosa'' === ===Background=== Saccharopolyspora spinosa is known for producing an insecticide, spinosad. Spinosad is a m..."

==Organism Page==

=== ''Saccharopolyspora spinosa'' ===

===Background===

Saccharopolyspora spinosa is known for producing an insecticide, spinosad. Spinosad is a mixture of chemical compounds in the spinosyn family.

===Growth & Maintenance===

=====Sporulation=====

 S. spinosa sporulates well on yeast malt extract agar plates.

===Strains===

===Links===
=====Genomes=====
=====Transcriptomes=====
=====Proteomics=====
=====Protocols=====

====Availability====
=====Culture Collections=====

Saccharopolyspora

2019-08-30T20:42:22Z

H Otani: Added two species

Saccharopolyspora is a genus of filamentous bacteria in the family Pseudonocardiaceae along with [[Amycolatopsis]] and [[Pseudonocardia]]. Best known species is Saccharopolyspora erythraea which makes the antibiotic [https://en.wikipedia.org/wiki/Erythromycin erythromycin].

[[Saccharopolyspora erythraea]] - known for producing erythromycin.

[[Saccharopolyspora spinosa]] - known for producing spinosyns.

YMPD

2019-08-30T20:37:13Z

H Otani: Created page with "=YMPD (Yeast Meat Peptone Dextrose)= For growth of ''Streptomyces griseus''. ==Preparation== Per litre: *2 g yeast extract *2.2 g meat extract *4 g Bacto peptone *5 g Na..."

=YMPD (Yeast Meat Peptone Dextrose)=

For growth of ''[[Streptomyces griseus]]''.

==Preparation==
Per litre:
*2 g yeast extract
*2.2 g meat extract
*4 g Bacto peptone
*5 g NaCl
*2 g magnesium sulphate heptahydrate (MgSO4·H2))
*1 g glucose
*1 L with distilled water

'''Instructions'''
#Dissolve ingredients in water
#pH 7.2
#For solid medium, add 22 g agar
#Autoclave

==Uses==

==Notes==

==References==
Ohnishi Y, et al. 2008. Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus IFO 13350. J. Bacteriol. 190: 4050-4060.

Media recipes

2019-08-30T20:20:40Z

H Otani: /* Media Recipes */ YMPD

{{DISPLAYTITLE:Media Recipes}}

[[File:Actinobase-icons-trans_Media-recipes.png|150px|Media Recipes|link=Media_recipes]]

=Media Recipes=

[[A1]]

[[CM]] (Complete medium)

[[DNA]] (Difco Nutrient Agar) | [[DNB]] (Difco Nutrient Broth) | [[SNA]] (Soft Nutrient Agar)

[[Emerson medium]]

[[Gause's No. 1]]

[[GYM]] (Glucose + Yeast Extract + Malt Extract)

[[HT]] (Hickey-Tresner)

[[HV]]

[[IMA]] (Smash)

[[LB]] (Lysogeny Broth) | [[L]] (L agar)

[[MC agar]] (Milk Casein Agar)

[[MM]] (Minimal medium)

[[SFM | MS]] (Mannitol Soya agar - a.k.a. Soya Flour Mannitol (SFM))

[[MYM]] (Maltose + Yeast Extract + Malt Extract)

[[NMMP]] (minimal liquid medium)

[[OT]] (oligotrophic agar)

[[PDA]] (Potato Dextrose Agar)

[[R2]] | [[R2YE]] | [[R5]]

[[SC]] (Starch Casein agar)

[[SFM]] (Soya Flour Mannitol - a.k.a. Mannitol Soya agar)

[[SMM]] (supplemented liquid minimal medium) | [[SMMS]] (supplemented minimal medium, solid)

[[SPY]] (Salt Peptone Yeast)

[[TSB]] (Tryptone Soy Broth)

[[YEME]] (Yeast Extract + Malt Extract)

[[YMPD]] (Yeast Meat Peptone Dextrose)

[[YP]] (Yeast Peptone) | [[YPD]] (Yeast Peptone Dextrose)

[[2X YT]]

====International ''Streptomyces'' Project Media (ISP's)====

[[ISP2]]

[[ISP3]]

[[ISP4]]

[[ISP5]]

[[ISP6]]

[[ISP7]]

==Media Supplements==

[[Antibiotic stocks and working concentrations]]

[[Indicator compounds]]

[[Media supplements for activating cryptic biosynthetic gene clusters]]

[[Media supplements for induction gene expression from inducible promoters]]

High GC PCR

2019-08-30T20:13:18Z

H Otani: Added betaine.

=High GC PCR=
Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.

==Tips for successful high GC PCR==

*Additives such as dimethyl sulfoxide (DMSO) or betaine can be added to reaction mixtures up to a final concentration of 1-10% or 0.5 M, respectively, which can help denature DNA and promote specificity.

*When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).

*When amplifying a region of DNA, try to find small regions of higher AT content that could form part of the primer sequence. This well help lower the Tm of the primers and reduce self-annealing and secondary structure formation. Try to aim for a primer with no more than 75% GC content.

*Some specific polymerases can aid high GC DNA amplification. High fidelity polymerases will help improve specificity.

*Q5® High-Fidelity DNA Polymerase comes with a high GC enhancer that can be added to reaction mixtures up to a final concentration of 10-20% to improve specificity.

==General protocol for ''Streptomyces'' colony PCR==
#Streak your strain of interest for single colonies on [[DNA]] medium.
#Pick a single colony and crush in 100µl 50% DMSO.
#Heat at 65ºC for ~20 minutes.
#Add 1µl of this to 10µl of PCR mix

Groups

2019-08-30T19:46:50Z

H Otani: Added some groups

Below is an alphabetical list of research groups working on Streptomyces, closely related actinomycete bacteria and their natural products. *Indicates membership of the international organising committee for ISBA the International Symposium on the Biology of Actinomycetes. If you'd like to be added please contact us via [https://twitter.com/ActinoBase @actinobase] or use the contact link in the menu bar.

====World Map of Research Groups====
<html>
<iframe src="https://www.google.co.uk/maps/d/u/0/embed?mid=14bRNmsDo2vIXP_I8CVOgW4DJGB7ewkuf" width="640" height="480"></iframe>
</html>

====Research Groups List====
[https://www.swansea.ac.uk/staff/medicine/learningandteaching/delsolabascaler/ Prof. Ricardo Del Sol Abascal]

[https://scholar.google.co.uk/citations?user=0TuurLQAAAAJ Prof. Bertrand Aigle]

[http://fcm.ens.uabc.mx/?p=personal.php&per=nmillan Prof. Natalie Millán Aguiñaga]

[https://langebio.cinvestav.mx/en/Dr-Francisco-Barona Dr. Paco Barona-Gomez] *

[https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/interfakultaere-einrichtungen/imit/arbeitsgruppen/mikrobiologie-biotechnologie/research-groups/bera/ Dr. Agnieszka Bera]

[https://www.kuleuven.be/wieiswie/en/person/00006977 Prof. Kristel Bernaerts]

[https://www.faculty.mun.ca/dbignell/ Dr. Dawn Bignell]

[https://sites.wustl.edu/blodgettlab/ Prof. Joshua Blodgett]

[https://www.unioviedo.es/IUOPA/?page_id=1852&lang=en Dr. Felipe Lombó Brugos]

[https://blogs.cornell.edu/buckley/ Prof. Dan Buckley]

[https://www.jic.ac.uk/people/mark-buttner/ Prof. Mark Buttner]

Prof. Beatriz Cámara

[https://scholar.google.com/citations?user=na_rzP4AAAAJ&hl=en Prof. Chang-Jun Cha]

[https://warwick.ac.uk/fac/sci/chemistry/research/challis/challisgroup Prof. Greg Challis]

[http://www.claessen-lab.nl/ Prof. Dennis Claessen]

[https://www.lerner.ccf.org/cms/claesen/ Prof. Jan Claesen]

[https://www.corre-lab.com/ Prof. Chris Corre]

[https://www.proimi.org.ar/miembros/dr-jose-sebastian-davila-costa Dr. José Sebastián Davila Costa]

[https://www.unimi.it/en/ugov/person/paolo-cortesi Prof. Paolo Cortesi]

[https://www.abdn.ac.uk/ncs/profiles/h.deng Dr. Hai Deng]

[http://skmml.sjtu.edu.cn/en/Faculty_Content.aspx?id=43 Prof. Zixin Deng] *

[http://publish.ucc.ie/researchprofiles/D021/adobson Prof. Alan Dobson]

Prof. Cristina Dorador

[http://www.naicons.com/about-us/management-team/stefano-donadio.html Dr. Stefano Donadio] *

[https://www.strath.ac.uk/staff/duncankatherinedr/ Dr. Kate Duncan]

[https://www.swansea.ac.uk/staff/medicine/learningandteaching/dysonpj/ Prof. Paul Dyson]

[https://www.abdn.ac.uk/ncs/people/ncs/profiles/r.ebel Dr. Rainer Ebel]

[https://fhs.mcmaster.ca/biochem/elliot_marie.html Prof. Marie Elliot]

[https://eustaquio.lab.uic.edu Dr. Alessandra Eustaquio]

[https://www.swansea.ac.uk/staff/medicine/research/faceyp/#publications=is-collapsed Dr. Paul Facey]

[http://scrippsscholars.ucsd.edu/wfenical Prof. William Fenical]

[http://www.uniovi.es/en/-/manteca-fernandez-angel Prof. Ángel Manteca Fernández]

[https://www.edgehill.ac.uk/biology/staff/lorena-fernandez-martinez/ Dr. Lorena Fernández-Martínez]

[https://www.biology.lu.se/klas-flardh Prof. Klas Flärdh]

[https://www.flinders.edu.au/people/chris.franco Prof. Chris Franco] *

[https://rjmg.wp.st-andrews.ac.uk/rebecca-goss/ Dr. Rebecca Goss]

[https://www.strath.ac.uk/staff/herronpauldr/ Dr. Paul Herron]

[https://appl-mol-microbiol-shinshu-u.jimdo.com/ Prof. Jun Hosaka]

[https://www.strath.ac.uk/staff/hoskissonpauldr/ Prof. Paul Hoskisson] *

[https://www.geomar.de/en/mitarbeiter/fb3/ms/uhentschel/ Prof. Ute Hentschel]

[https://people.uea.ac.uk/m_hutchings Prof. Matt Hutchings]

[https://www.kitasato-u.ac.jp/lisci/english/kitasato-LIS/ILS-Lab21en.html Prof. Hauro Ikeda]

[http://www.ibmb.uni.wroc.pl/zmm/jakimowicz.html Prof. Dagmara Jakimowicz]

[https://www.abdn.ac.uk/ncs/profiles/m.jaspars/ Prof. Marcel Jaspars]

[http://scrippsscholars.ucsd.edu/pjensen Prof. Paul Jensen]

Prof. Weihong Jiang

[https://plpa.cfans.umn.edu/people/faculty/linda-kinkel Prof. Linda Kinkel]

[https://imbm.co.za/staff/dr-bronywn-kirby/ Dr. Bronwyn Kirby-McCullough]

[https://people.uea.ac.uk/g_kelemen Dr. Gabriela Kelemen]

[https://www.sav.sk/?lang=en&doc=user-org-user&user_no=1318 Dr. Ján Kormanec]

Dr. Sylvie Lautru

[https://scholar.google.co.uk/citations?user=yL6B0g8AAAAJ Prof. Pierre Leblonde]

[https://scholar.google.com/citations?user=L-f_SkkAAAAJ&hl=en Prof. Ahmed Lebrihi]

[http://loesgenlab.org/ Prof. Sandra Loesgen]

[https://pppmb.cals.cornell.edu/people/rosemary-loria/ Prof. Rose Loria] *

Prof. Yinhua Lu

Prof. Luis Maldonado

[https://www.dsmz.de/research/bioresources-for-bioeconomy-and-health-research/ Prof. Yvonne Mast]

[https://www.duq.edu/academics/faculty/joseph-mccormick Prof. Joe McCormick]

[https://biofun.uniovi.es/areas/microbiologia/-/asset_publisher/0009/content/mendez-fernandez-m-del-carmen?redirect=%2Fareas%2Fmicrobiologia Prof. Carmen Méndez]

[https://chemistry.illinois.edu/douglasm Prof. Douglas Mitchell]

[http://scrippsscholars.ucsd.edu/bsmoore/pages/moore-lab-research Prof. Bradley Moore]

[https://jgi.doe.gov/about-us/organization/strategic-management/nigel-mouncey/ Dr. Nigel Mouncey]

[https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/interfakultaere-einrichtungen/imit/arbeitsgruppen/mikrobiologie-biotechnologie/research-groups/musiol-kroll/ Dr. Ewa Musiol-Kroll]

[https://uni-tuebingen.de/en/faculties/faculty-of-science/departments/interfaculty-facilities/imit/research-groups/microbiology-biotechnology/research-groups/muth/ Dr. Gunter Muth]

[http://biochemistry.utoronto.ca/person/justin-r-nodwell/ Prof. Justin Nodwell] *

[http://park.itc.u-tokyo.ac.jp/hakko/ Prof. Yasuo Ohnishi]

[http://www.sussex.ac.uk/profiles/117009 Dr. Mark Paget]

[https://micro.biol.ethz.ch/research/piel.html Prof. Jorn Piel]

[https://www.unipa.it/persone/docenti/p/a.maria.puglia Prof. Anna Maria Puglia]

[https://scholar.google.com/citations?user=IDzDl10AAAAJ&hl=en Dr. Shanti Ratnakomala]

[http://labos.ulg.ac.be/cip/research/streptomyces-genetics-and-development/ Dr. Sébastien Rigali]

[http://biosci.snu.ac.kr/en/people/faculty?mode=view&profidx=13 Prof. Jung-Hye Roe] *

[http://cput.academia.edu/MarilizeLeRoesHill Dr. Marilize Le Roes-Hill]

[https://www.surrey.ac.uk/people/claudio-avignone-rossa-frsb Prof. Claudio Avignone Rossa FRSB]

Prof. José Salas

Prof. Richard Samuels

[https://pharmacy.uic.edu/profiles/sanchelm/ Dr. Laura Sanchez]

[https://www.northumbria.ac.uk/about-us/our-staff/s/dr-vartul-sangal/ Dr. Vartul Sangal]

[https://www.biologie.uni-halle.de/microbiology/sawers/ Prof. Gary Sawers]

[https://www.uni-osnabrueck.de/universitaet/personensuche/personendetails.html?config_id=182d4abe417c9851aacad4acd6e0df1b&module=TemplatePersondetails&range_id=studip&target=15852&username=hschremp Prof. Hildgund Schrempf]

[https://www.schlimpertlab.com Dr. Susan Schlimpert]

[https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/134/dr-ryan-f-seipke Dr. Ryan Seipke]

[https://www.scripps.edu/shen/ Prof. Ben Shen]

[https://www.lsi.umich.edu/profiles/david-sherman-phd Prof. David Sherman]

[https://www.york.ac.uk/biology/research/molecular-microbiology/maggie-smith/ Prof. Maggie Smith]

[https://research.brighton.ac.uk/en/persons/colin-smith Prof. Colin Smith]

[https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/interfakultaere-institute-und-zentren/imit/arbeitsgruppen/mikrobiologie-biotechnologie/research-groups/stegmann/ Dr. Evi Stegmann]

[https://biochemistry.tamu.edu/people/straight-paul/ Dr. Paul Straight]

[https://www.northumbria.ac.uk/about-us/our-staff/s/professor-iain-sutcliffe/ Prof. Iain Sutcliffe]

[https://www.faculty.mun.ca/ktahlan/ Dr. Kapil Tahlan]

[https://www.research.manchester.ac.uk/portal/eriko.takano.html Prof. Eriko Takano]

[http://www.riken.jp/en/research/labs/csrs/riken_kribb/ Dr. Shunji Takahashi]

[https://www.biologie.hu-berlin.de/de/gruppenseiten/mikrobiologie/tschowri_group Dr. Natalia Tschowri]

[https://scholar.google.co.th/citations?user=xj8zuvoAAAAJ&hl=th Prof. Arinthip Thamchaipenet] *

[https://people.csiro.au/T/L/Louise-Thatcher Dr. Louise Thatcher]

[https://plantandmicrobiology.berkeley.edu/profile/traxler Dr. Matt Traxler]

[https://scholar.google.es/citations?user=t4UxSxEAAAAJ&hl=en Dr. Martha Trujillo] *

[https://www.jic.ac.uk/people/andrew-truman/ Dr. Andy Truman]

Dr. Kenji Ueda *

[https://chemistry.illinois.edu/vddonk Prof. Wilfred van der Donk]

[https://www.swansea.ac.uk/staff/medicine/learningandteaching/vankeuleng/ Dr. Geertje van Keulen]

[https://www.universiteitleiden.nl/en/staffmembers/gilles-van-wezel#tab-1 Prof. Gilles van Wezel] *

Prof. Marie-Joelle Virolle

[https://www.irb.hr/eng/About-RBI/People/Dusica-Vujaklija Dr. Dusica Vujaklija]

[http://www.fau.edu/hboi/mbbr/biosynth-1.php Prof. Guojun Wang]

[https://www.dtu.dk/english/service/phonebook/person?id=91195&tab=2&qt=dtupublicationquery Prof. Tilmann Weber]

[https://warwick.ac.uk/fac/sci/lifesci/people/ewellington/ Prof. Liz Wellington] *

[https://www.helmholtz-hzi.de/en/research/research-topics/anti-infectives/microbial-strain-collection/our-research/#anchorsection Prof. Joachim Wink]

[https://www.jic.ac.uk/people/professor-barrie-wilkinson/ Prof. Barrie Wilkinson]

[https://www.bioscience.utah.edu/faculty/winter/ Prof. Jaclyn Winter]

[https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/interfakultaere-institute-und-zentren/imit/arbeitsgruppen/mikrobiologie-biotechnologie/ Prof. Wolfgang Wohlleben] *

[http://www.thewrightlab.com Prof. Gerry Wright]

[https://www.ewha.ac.kr/mbs/ewhakr/jsp/sprofile/sprofile_View.jsp?id=ewhaen_030103030100&cateId=80&sabun=MTUwODYx&tab=TAB2 Prof. Yeo-Joon Yoon]

[http://www.ibmb.uni.wroc.pl/zmm/zakrzewska.html Dr. Jolanta Zakrzewska-Czerwińska]

[http://zhanglab.caspmi.cn/?people/current-members/zhanglixin.html Prof. Lixin Zhang] *

[http://faculty.scs.illinois.edu/~zhaogrp/ Prof. Huimin Zhao]

[https://uni-tuebingen.de/en/faculties/faculty-of-science/departments/interfaculty-institutes-and-centres/imit/research-groups/applied-natural-products-genome-mining/ Prof. Nadine Ziemert]

[https://uni-tuebingen.de/en/faculties/faculty-of-science/departments/pharmacy-and-biochemistry/pharmazie/pharmaceutical-biology/ak-gust/ Dr. Bertolt Gust]

[https://uni-tuebingen.de/en/faculties/faculty-of-science/departments/pharmacy-and-biochemistry/pharmazie/pharmaceutical-biology/ak-kaysser/ Prof. Leonard Kaysser]

[http://www.im.ac.cn/english1/pi/tanhuarong.htm Prof. Huarong Tan]

Sequence databases

2019-08-30T19:20:50Z

H Otani: Added IMG/M and Genome Portal

[https://www.embl.de EMBL]

[https://www.ncbi.nlm.nih.gov NCBI]

[https://img.jgi.doe.gov/ IMG/M]

[https://genome.jgi.doe.gov/portal/ JGI Genome Portal]