Team:Paris Bettencourt/YonatanTest
From 2013.igem.org
![](https://static.igem.org/mediawiki/2013/d/d9/PB_achievementsbanenr.png)
Detect
Background
CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.
Aims
Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.
Results
- Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks.
- Testing the new assembly standard for our cloning.
![](https://static.igem.org/mediawiki/2013/5/55/PB_Bb_target.png)
![](https://static.igem.org/mediawiki/2013/9/99/PB_fig_target.png)
CRISPR anti-Kan plasmids target kanamycin resistant E. coli. WT (blue bars) and a kanamycin resistant strain (KanR, red bars) were co-transformed with a plasmid carrying the Cas9 construct, and a second plasmid carrying the anti-Kanamycin gRNA. WT E. coli was transformed with one and both plasmids. KanR E. coli couldn’t be tranformed with both plasmids because of Cas9-induced cleavage of the chromosome specifically at the KanR cassette, with about 99% efficiency.