e coli s17  (Addgene inc)

Journal: Poultry Science

Article Title: Development of the CRISPR/Cas9 system for genome editing in Riemerella anatipestifer

doi: 10.1016/j.psj.2025.105696

Figure Lengend Snippet: Construction and characteristics of CRISPR/Cas9 based genome-editing plasmid pCasRA-SacB in R. anatipestifer . pRA0726 ori and p15A ori, the replicon used in R. anatipestifer and E. coli , respectively; high-expression promoter, the promoter of B739_0921 gene, driving the expression of Cas9; rpsL promoter, the promoter of rpsL gene, driving the expression of sgRNA; CmR and cfxA, the chloramphenicol and cefoxitin resistance marker used in E. coli and R. anatipestifer , respectively; BsaI and SalI, the site used to clone of sgRNA and homologous arms; sacB and oriT, for plasmid curing and transfer of plasmid via conjugation.

Article Snippet: E. coli S17-1 as the donor to introduce the resulting plasmid into R. anatipestifer ATCC 11845 by conjugation experiments.

Techniques: CRISPR, Plasmid Preparation, Expressing, Marker, Conjugation Assay

Journal: Microbiology Spectrum

Article Title: Markerless mutagenesis enables isoleucine biosynthesis solely from threonine in Methanothermobacter marburgensis

doi: 10.1128/spectrum.03068-24

Figure Lengend Snippet: PCR analysis of positive and negative selection for a pure M. marburgensis Δ hpt strain. ( A ) Depiction of the first merodiploid state of M. marburgensis with E. coli vector genome integrated with a single upstream homologous recombination event. II represents an inside-inside hpt vector primer combination for a PCR fragment. EO represents a primer combination specific for the E. coli vector backbone and native genomic DNA of M. marburgensis . ( B ) First analysis after negative selection with 8-azahypoxanthine with a primer combination outside of the hpt homologous flanking regions on genomic DNA of M. marburgensis (OO). While clones 1, 3, and 4 still show both the wild-type and deletion signals, the clone 2 signal points toward clean deletion of hpt . ( C ) PCR analysis of genomic DNA from M. marburgensis Δ hpt after a dilution series of 1:10,000 and 1:1,000,000 resulting in the final M. marburgensis Δ hpt pure strain. Abbreviations for primer combinations are the same across ( A–C ) and explained in panel ( D ). ( D ) Schematic drawing of genomic DNA states in wild-type M. marburgensis , M. marburgensis merodiploid, and M. marburgensis Δ hpt . Arrows and characters match the letters used in A–C and act as references for PCR fragment lengths.

Article Snippet: We purchased E. coli S17-1 (DSM 9097) from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) for conjugal transfer of DNA.

Techniques: Selection, Plasmid Preparation, Homologous Recombination, Clone Assay