Team:Paris Bettencourt/Results

CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.

MycoSIR E. coli depend on our synthetic pathway for growth. E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth

TDMH expression kills mycobacteria in culture. We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.

Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.