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broad host range conjugative plasmid prk24  (Addgene inc)


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    Addgene inc broad host range conjugative plasmid prk24
    Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via <t>pRK24-mediated</t> conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.
    Broad Host Range Conjugative Plasmid Prk24, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/broad+host+range+conjugative+plasmid+prk24/bio_rxiv__64898__2026__03__21__713436-246-1-5?v=Addgene+inc
    Average 92 stars, based on 13 article reviews
    broad host range conjugative plasmid prk24 - by Bioz Stars, 2026-08
    92/100 stars

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    1) Product Images from "Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems"

    Article Title: Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems

    Journal: bioRxiv

    doi: 10.64898/2026.03.21.713436

    Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.
    Figure Legend Snippet: Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.

    Techniques Used: Plasmid Preparation, Modification, Methylation, Produced, Conjugation Assay, CRISPR, Sequencing, Homologous Recombination, Expressing, Biomarker Discovery, Generated, Transformation Assay, Electroporation



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    broad host range conjugative plasmid prk24  (Addgene inc)
    92
    Addgene inc broad host range conjugative plasmid prk24
    Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via <t>pRK24-mediated</t> conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.
    Broad Host Range Conjugative Plasmid Prk24, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/broad+host+range+conjugative+plasmid+prk24/bio_rxiv__64898__2026__03__21__713436-246-1-5?v=Addgene+inc
    Average 92 stars, based on 1 article reviews
    broad host range conjugative plasmid prk24 - by Bioz Stars, 2026-08
    92/100 stars
    		  Buy from Supplier

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    Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.

    Journal: bioRxiv

    Article Title: Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems

    doi: 10.64898/2026.03.21.713436

    Figure Lengend Snippet: Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.

    Article Snippet: The broad-host-range conjugative plasmid pRK24 (Addgene plasmid # 51950, courtesy of Farren Isaacs) was first introduced into E. coli MC variants and selected on tetracycline (10 μg/mL).

    Techniques: Plasmid Preparation, Modification, Methylation, Produced, Conjugation Assay, CRISPR, Sequencing, Homologous Recombination, Expressing, Biomarker Discovery, Generated, Transformation Assay, Electroporation