Based on the fundamental synthetic biology methods, include the molecular cloning, long PCR, recombinant construction, restriction digestion and yeast transformation, our project aims to enable Saccharomyces cerevisiae to resist viruses utilizing the CRISPR system found in most bacteria and archaea lately.
To ensure the accurate output, we divide the original linear gene into several elements , and also purify and propagate the complete target sequence to make the both two models work in the host cell. To present the actual effect of CRISPR in yeast, we design our own mimic virus construction based on the homologous sequences shown in the specific viral genome database, which can be recognized, and control the baseline both for anti-virus ability and CRISPR's effect in its original form.
More specifically, we first obtain our CRISPR system by genome-extraction from E.coli str W3110, and clone the respective target genes as modules. The construction, as well as the mimic virus construction, IS made by restriction-ligase way. We transform the mimic virus plasmid first by lithium acetate method and verify the availability by several antibiotics. We then import CRISPR system in the same way, utilizing the Ochre mutation and GFP switch-off to evaluate the anti-virus feasibility.
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And CRISPR-based defense system can also protects them against the invading DNA and/or RNA elements. It is believed that the integrated CRISPR sequences have the ability to form a genetic memory which prevents the host from being infected. The CRISPRs and Cas (CRISPR-associated) interact and form this prokaryotic adaptive immune system....
