Lysis of shiga toxin-producing Escherichia coli by delivery of Crispr/Cas9 cascade coding cosmid targeting Stx 1 and Stx 2 genes via lambda phage delivery.

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Lysis of shiga toxin-producing Escherichia coli by delivery of Crispr/Cas9 cascade coding cosmid targeting Stx 1 and Stx 2 genes via lambda phage delivery system Amanda Rutledge

How E. coli become Shiga toxin-producing Shiga toxin-encoding phage (such as 933W) Become shiga toxin producing when undergo lysogenesis with shiga toxin encoding phage Escherichia coli

How E. coli become Shiga toxin-producing Shiga toxin-encoding phage (such as 933W) Escherichia coli

How E. coli become Shiga toxin-producing Escherichia coli

What We’ll Need… Modified Lambda Phage (CY1591) Plasmid pUC18 kanamycin resistance gene inside cos site Plasmid pUC18 functioning cos site shiga-toxin targeting Crispr/Cas Tet promoters Plasmid pACYC184 Tet repressor protein gene E. Coli Y1088 strain hsdM+

Modified Lambda Phage (Cronan 2003) Cronan JE. Cosmid-Based System for Transient Expression and Absolute Off-to-On Transcriptional Control of Escherichia coli Genes. Journal of Bacteriology. 2003;185(22):6522-6529. doi:10.1128/JB.185.22.6522-6529.2003.

Plasmid pUC18 Tet P cas3 Tet P casA-E Repeat S1 Repeat S2 Repeat pUC18 Cos site

Plasmid pACYC184 Hind III Tet repressor AvaI Hind III AvaI

Testing for proper packaging of cosmid

What can these phage do? Crispr/Cas9

Questions?