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Team:GeorgiaTech/Future
From 2011.igem.org
The ultimate goal of our project was to utilize this system as a mobile gene targeting system capable of eliminating antibiotic resistance. The protoype construct, pSTINGER, was designed for quantitative modeling, proof of concept, and vaccination on a single plasmid. The premise of our vaccine is that it is able to conjugate within a population and therefore an origin of transfer consistent with most conjugation mechanisms [citation] (OriT) was included. In total, the construct contains one functional gene, and that gene is Cas9. Cas9 originates from the CRISPR/Cas system of Streptococcus thermophilus LMD-9 and serves as open analog to the functionally evidenced CRISPR3 locus within commercial strain Streptococcus thermophilus DGCC7710. Cas9 requires a highly stable and characterized promoter, so a Sigma factor 70 sequence was included in the construct. Cas9 has been shown as the sole Cas gene required for CRISPR RNA directed cleaving of recognized DNA in mobile constructs also containing native CRISPR DNA[citation].
In order to explicitly target DNA of interest we designed a synthetic Spacer repeat region flanked by the native leader and terminating sequence from LMD-9. Each spacer sequence of numbers 1 through 5 is a 30 base pair region with in the active functional domain of the translated genes. For functional proof of concept we included a kanamycin resistance gene to demonstrate function loss of the resistance phenotype. For quantitative modeling, we envisioned a continous flow cytometer for monitoring population containing a GFP coding target plasmid, and pSTINGER with RFP cloned in. In this experiment, we seek quantitative flourescence data to compliment our population model detailed elsewhere. Lastly we seek to go for the throat, we target functional domains of New Delhi metallo-beta-lactamase 1, vanA-type vancomycin resistance genes, and MecA-type methicillin resistance genes for downstream experiments.
