Part:BBa_K3409013:Experience

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Applications of BBa_K3409013

In order to validate our system, we will perform an Agar Diffusion Assay. This will confirm the proper secretion of McpM as well as its lytic activity on the target. For this, a sensitive strain is plated (E. coli DH5α or BL21) and disks are inoculated with different bacterial cultures. The positive control is E. coli DH10B containing the native and functional MccPDI gene cluster in a plasmid, kindly provided by Mr. Call. We should see a zone of inhibition around the disk as the microcin is secreted and kills the surrounding cells. This control will be compared to our engineered bacteria: E. coli DH5α containing the reconstructed MccPDI cluster. The negative control is plating the dish with E. coli ΔOmpF which is known to resist against the Microcin’s action thanks to its mutation.

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