dsm 10 type strain  (DSMZ)

Journal: Microbiology Resource Announcements

Article Title: Draft Genome Sequence of the Type Strain Bacillus subtilis subsp. subtilis DSM10

doi: 10.1128/MRA.00158-21

Figure Lengend Snippet: Sequence variations between B. subtilis DSM10 T and NCIB3610

Article Snippet: The Bacillus subtilis subsp. subtilis type strain DSM10 is a generally accessible Bacillus strain from the German Collection of Microorganisms and Cultures GmbH (DSMZ).

Techniques: Sequencing, Mutagenesis, Membrane

Journal: Applied and Environmental Microbiology

Article Title: A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792

doi: 10.1128/AEM.00408-20

Figure Lengend Snippet: Easy reprogramming of the two-plasmid CRISPR-Cas9 genetic tool for multiple genome editing. (A) Retargeting of the pGRNA plasmid by cloning two hybridized primers between the aTc-inducible promoter Pcm-2tetO1 and the gRNA scaffold. (B) Insertion of the editing template within the pGRNA plasmid by restriction enzyme-based cloning. (C) Iterative genome modification. Step 1, the strain is transformed with pCas9acr and transformants are selected on erythromycin-containing medium. Step 2, the resulting strain is transformed with a pGRNA editing plasmid, and transformants are selected on medium containing erythromycin, thiamphenicol, and lactose to activate the transcription of acrIIA4. Step 3, expression of cas9 is triggered on medium containing erythromycin and thiamphenicol and devoid of lactose so that transcription of acrIIA4 is no longer induced. The medium contains aTc to trigger the transcription of cas9 to select mutants in which the editing event occurred. Step 4, once aTc-resistant colonies have been confirmed, pGRNA is curated on medium containing erythromycin and no thiamphenicol, yielding cells ready for a new round of modification. ori, replication origin for E. coli; pIP404 and repH, compatible replication origins for C. acetobutylicum; catP, thiamphenicol-chloramphenicol resistance gene; ermB, erythromycin resistance gene; RS, restriction site; RE, restriction enzyme; DSB, double-strand break; HR, homologous recombination.

Article Snippet: Escherichia coli was grown aerobically at 37°C and 200 rpm in liquid LB medium or solid LB with 1.5% agar supplemented with erythromycin (500 μg ml −1 for solid medium and 100 μg ml −1 for liquid medium), chloramphenicol (25 μg ml −1 for solid medium and 12.5 μg ml −1 for liquid medium), or tetracycline (20 μg ml −1 ) if necessary. table ft1 table-wrap mode="anchored" t5 TABLE 4 caption a7 Bacterial strain or plasmid Relevant characteristics Source or reference Strains C. acetobutylicum DSM 792 Wild type DSMZ E. coli NEB 10-beta Cloning strain NEB Plasmids pAN2 tetA , Φ3T I gene, p15A origin 5 , 30 pFW01 ermB , ColE1 origin, pCB102 origin 16 pCas9 ind ermB , ColE1 origin, pCB102 origin, cas9 (Pcm-tetO2/1 promoter), tetR 16 pCas9 acr pCas9 ind derivative with acrIIA4 (P bgal promoter) and bgaR insertions This study pEC750C catP , ColE1 origin, pIP404 origin 16 pGRNA ind pEC750C derivative with gRNA expression cassette (Pcm-2tetO1 promoter) insertion This study pGRNA- bdhA pGRNA ind derivative targeting bdhA This study pGRNA-Δ bdhA pGRNA- bdhA derivative with Δ bdhA editing template insertion This study pGRNA- bdhB pGRNA ind derivative targeting bdhB This study pGRNA-Δ bdhB pGRNA- bdhB derivative with Δ bdhB editing template insertion This study pGRNA-Δ bdhA Δ bdhB pGRNA- bdhB derivative with Δ bdhA Δ bdhB editing template insertion This study pGRNA- bdhC pGRNA ind derivative targeting bdhC This study pGRNA-Δ bdhC pGRNA- bdhC derivative with Δ bdhC editing template insertion This study pFW01- bdhA pFW01 derivative with bdhA insertion This study pFW01- bdhB pFW01 derivative with bdhB insertion This study pFW01- bdhC pFW01 derivative with bdhC insertion This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Plasmid Preparation, CRISPR, Clone Assay, Modification, Transformation Assay, Expressing, Homologous Recombination

Journal: Applied and Environmental Microbiology

Article Title: A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792

doi: 10.1128/AEM.00408-20

Figure Lengend Snippet: Relative transformation efficiencies of pGRNA template plasmids in DSM 792 containing pCas9ind or pCas9acr. Results shown are average values ± standard deviations from at least four independent replicates for each transformation. Absolute transformation efficiencies for pEC750C in DSM 792 (pCas9ind) and DSM 792 (pCas9acr) were 148 ± 37 CFU μg−1 and 121 ± 92 CFU μg−1, respectively.

Article Snippet: Escherichia coli was grown aerobically at 37°C and 200 rpm in liquid LB medium or solid LB with 1.5% agar supplemented with erythromycin (500 μg ml −1 for solid medium and 100 μg ml −1 for liquid medium), chloramphenicol (25 μg ml −1 for solid medium and 12.5 μg ml −1 for liquid medium), or tetracycline (20 μg ml −1 ) if necessary. table ft1 table-wrap mode="anchored" t5 TABLE 4 caption a7 Bacterial strain or plasmid Relevant characteristics Source or reference Strains C. acetobutylicum DSM 792 Wild type DSMZ E. coli NEB 10-beta Cloning strain NEB Plasmids pAN2 tetA , Φ3T I gene, p15A origin 5 , 30 pFW01 ermB , ColE1 origin, pCB102 origin 16 pCas9 ind ermB , ColE1 origin, pCB102 origin, cas9 (Pcm-tetO2/1 promoter), tetR 16 pCas9 acr pCas9 ind derivative with acrIIA4 (P bgal promoter) and bgaR insertions This study pEC750C catP , ColE1 origin, pIP404 origin 16 pGRNA ind pEC750C derivative with gRNA expression cassette (Pcm-2tetO1 promoter) insertion This study pGRNA- bdhA pGRNA ind derivative targeting bdhA This study pGRNA-Δ bdhA pGRNA- bdhA derivative with Δ bdhA editing template insertion This study pGRNA- bdhB pGRNA ind derivative targeting bdhB This study pGRNA-Δ bdhB pGRNA- bdhB derivative with Δ bdhB editing template insertion This study pGRNA-Δ bdhA Δ bdhB pGRNA- bdhB derivative with Δ bdhA Δ bdhB editing template insertion This study pGRNA- bdhC pGRNA ind derivative targeting bdhC This study pGRNA-Δ bdhC pGRNA- bdhC derivative with Δ bdhC editing template insertion This study pFW01- bdhA pFW01 derivative with bdhA insertion This study pFW01- bdhB pFW01 derivative with bdhB insertion This study pFW01- bdhC pFW01 derivative with bdhC insertion This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Transformation Assay

Journal: Applied and Environmental Microbiology

Article Title: A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792

doi: 10.1128/AEM.00408-20

Figure Lengend Snippet: CRISPR-Cas9 strategy and PCR analyses of representative deletion mutants constructed in this study. (A and B) bdh loci from DSM 792. Genes are shown in green, gRNA target sites are indicated in violet, and homology sequenced between gDNA and the editing templates is in blue. (A) bdhA-bdhB locus. (B) bdhC locus. (C) Amplification of the bdhA-bdhB and bdhC loci. Amplification with primer pair P30-P31 yields a PCR product of 5,173, 4,027, 4,024, or 2,578 bp in wild-type (WT), ΔbdhA, ΔbdhB, or ΔbdhA ΔbdhB strains, respectively. Amplification with primer pair P32-P33 yields a PCR product of 3,124 bp or 1,993 bp in WT or ΔbdhC strains, respectively. Lane M, 2-log DNA ladder (0.1 to 10 kb; NEB); lane H2O, amplification on water.

Article Snippet: Escherichia coli was grown aerobically at 37°C and 200 rpm in liquid LB medium or solid LB with 1.5% agar supplemented with erythromycin (500 μg ml −1 for solid medium and 100 μg ml −1 for liquid medium), chloramphenicol (25 μg ml −1 for solid medium and 12.5 μg ml −1 for liquid medium), or tetracycline (20 μg ml −1 ) if necessary. table ft1 table-wrap mode="anchored" t5 TABLE 4 caption a7 Bacterial strain or plasmid Relevant characteristics Source or reference Strains C. acetobutylicum DSM 792 Wild type DSMZ E. coli NEB 10-beta Cloning strain NEB Plasmids pAN2 tetA , Φ3T I gene, p15A origin 5 , 30 pFW01 ermB , ColE1 origin, pCB102 origin 16 pCas9 ind ermB , ColE1 origin, pCB102 origin, cas9 (Pcm-tetO2/1 promoter), tetR 16 pCas9 acr pCas9 ind derivative with acrIIA4 (P bgal promoter) and bgaR insertions This study pEC750C catP , ColE1 origin, pIP404 origin 16 pGRNA ind pEC750C derivative with gRNA expression cassette (Pcm-2tetO1 promoter) insertion This study pGRNA- bdhA pGRNA ind derivative targeting bdhA This study pGRNA-Δ bdhA pGRNA- bdhA derivative with Δ bdhA editing template insertion This study pGRNA- bdhB pGRNA ind derivative targeting bdhB This study pGRNA-Δ bdhB pGRNA- bdhB derivative with Δ bdhB editing template insertion This study pGRNA-Δ bdhA Δ bdhB pGRNA- bdhB derivative with Δ bdhA Δ bdhB editing template insertion This study pGRNA- bdhC pGRNA ind derivative targeting bdhC This study pGRNA-Δ bdhC pGRNA- bdhC derivative with Δ bdhC editing template insertion This study pFW01- bdhA pFW01 derivative with bdhA insertion This study pFW01- bdhB pFW01 derivative with bdhB insertion This study pFW01- bdhC pFW01 derivative with bdhC insertion This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: CRISPR, Construct, Amplification

Journal: Applied and Environmental Microbiology

Article Title: A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792

doi: 10.1128/AEM.00408-20

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Escherichia coli was grown aerobically at 37°C and 200 rpm in liquid LB medium or solid LB with 1.5% agar supplemented with erythromycin (500 μg ml −1 for solid medium and 100 μg ml −1 for liquid medium), chloramphenicol (25 μg ml −1 for solid medium and 12.5 μg ml −1 for liquid medium), or tetracycline (20 μg ml −1 ) if necessary. table ft1 table-wrap mode="anchored" t5 TABLE 4 caption a7 Bacterial strain or plasmid Relevant characteristics Source or reference Strains C. acetobutylicum DSM 792 Wild type DSMZ E. coli NEB 10-beta Cloning strain NEB Plasmids pAN2 tetA , Φ3T I gene, p15A origin 5 , 30 pFW01 ermB , ColE1 origin, pCB102 origin 16 pCas9 ind ermB , ColE1 origin, pCB102 origin, cas9 (Pcm-tetO2/1 promoter), tetR 16 pCas9 acr pCas9 ind derivative with acrIIA4 (P bgal promoter) and bgaR insertions This study pEC750C catP , ColE1 origin, pIP404 origin 16 pGRNA ind pEC750C derivative with gRNA expression cassette (Pcm-2tetO1 promoter) insertion This study pGRNA- bdhA pGRNA ind derivative targeting bdhA This study pGRNA-Δ bdhA pGRNA- bdhA derivative with Δ bdhA editing template insertion This study pGRNA- bdhB pGRNA ind derivative targeting bdhB This study pGRNA-Δ bdhB pGRNA- bdhB derivative with Δ bdhB editing template insertion This study pGRNA-Δ bdhA Δ bdhB pGRNA- bdhB derivative with Δ bdhA Δ bdhB editing template insertion This study pGRNA- bdhC pGRNA ind derivative targeting bdhC This study pGRNA-Δ bdhC pGRNA- bdhC derivative with Δ bdhC editing template insertion This study pFW01- bdhA pFW01 derivative with bdhA insertion This study pFW01- bdhB pFW01 derivative with bdhB insertion This study pFW01- bdhC pFW01 derivative with bdhC insertion This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Plasmid Preparation, Clone Assay, Expressing