Part:BBa_K786031:Experience

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Expected Result

Firstly, we need to test if the proposed system present in the E. coli. We Used primers to amplify the Cas3 and cascade proteins from E. coli K12 genome DNA. Expected band size of cas3 2.7kbp and cascade 4.4kbp are shown in the gel photo, and sequencing has be done, which proves the existence of these proteins in its genome, thus we can utilize this CRISPR/Cas3 system as a tool for our new safety approach. In the future we can make them into biobrick format and apply into microorganism other than E. coli.

To evaluate whether the RSR system transformed into E. coli can degrade genomic DNA and eventually lead to cell death, we measured OD600 of cell cultures every 0.5 h and plotted the growth curves of transformed E. coli (indicated as solid lines) with or without 0.4 mM IPTG activation. Paired t-test showed that there is no significant difference in OD600 throughput the whole experiment with or without RSR activation by IPTG.

The result agrees with the percentage of survival (indicated as dashed lines) derived from live/dead fluorescent staining assay (Invitrogen) measuring bacterial apoptotic-like rate, which showed no difference in survival percentage with or without RSR activation. It was expected that the survival percentage drops significantly after IPTG induction. Further optimization of design of the RSR region and experimental conditions is required for more specific targeting.

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