cole1 origin  (DSMZ)

Journal: bioRxiv

Article Title: Generating functional plasmid origins with OriGen

doi: 10.1101/2025.02.03.636306

Figure Lengend Snippet: a , Experimental workflow for validating generated origins. b , Assessment of ColE1-type origin function in E. coli . Matched random sequences contain the same number of mutations relative to wild-type as their corresponding OriGen-generated sequences. Lighter green indicates a single nucleotide polymorphism or single nucleotide indel was detected after sequencing. Stars represent unsuccessful experiments, which are in the process of being rerun . c , RNAI secondary structure comparison of one OriGen-generated ColE1 example vs closest WT vs matched random mutant control. d , Assessment of uncharacterized Rep3-type origin function in E. coli . e , Distribution of known motifs in examples of Rep3 OriGen-generated origins vs closest WT vs matched random mutant. In both examples, the model-generated origins led to replication while the random mutants did not.

Article Snippet: Golden Gate assemblies were performed using type IIS restriction enzymes BsaI-HF®v2 (New England Biolabs, Cat. No. R3733S) for ColE1 origins and SapI (New England Biolabs, Cat. No. R0569S) for rep-associated origins.

Techniques: Generated, Sequencing, Comparison, Mutagenesis, Control

Journal: bioRxiv

Article Title: Generating functional plasmid origins with OriGen

doi: 10.1101/2025.02.03.636306

Figure Lengend Snippet: a , Experimental workflow for validating generated origins. b , Assessment of ColE1-type origin function in E. coli . Matched random sequences contain the same number of mutations relative to wild-type as their corresponding OriGen-generated sequences. Lighter green indicates a single nucleotide polymorphism or single nucleotide indel was detected after sequencing. Stars represent unsuccessful experiments, which are in the process of being rerun . c , RNAI secondary structure comparison of one OriGen-generated ColE1 example vs closest WT vs matched random mutant control. d , Assessment of uncharacterized Rep3-type origin function in E. coli . e , Distribution of known motifs in examples of Rep3 OriGen-generated origins vs closest WT vs matched random mutant. In both examples, the model-generated origins led to replication while the random mutants did not.

Article Snippet: ColE1 origins were synthesized as gene fragments (Twist Biosciences) containing BsaI sites and complementary overhangs (Supplemental Table 3).

Techniques: Generated, Sequencing, Comparison, Mutagenesis, Control

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