As a research team, our goals are two fold: to mobilize the crispr/cas bacterial immune system on a plasmid, and subsequently utilize this system as an intelligent gene targeting method capable of eliminating antibiotic resistance.
Proof of Concept
Streptococcus thermophilus is a gram positive bacteria with a highly active crispr system that is well characterized. We begin by showing that the Streptococcus thermophilus crispr system cleaves the kanamycin resistance gene, KanR, held on a vector. We determine crispr attributed kanamycin susceptibility by replica plating using an automated colony picking and plate inoculation apparatus. Plates are screened for optical density and logically indexed to determine occurrence of kanamycin susceptibility. The crispr system is an adaptive immune system, and recognizable motifs of foreign DNA are subsequently inserted into the crispr locus of the chromosome for future recognition. Susceptible colonies from the originating plate are then isolated and the adapted crispr system is cloned into a new vector. In a crispr deficient strain (E. coli or S. thermophilus), the crispr vector and the kanamycin vector is introduced, and the transformed cells are screened for functionality using the method previously described.
"
