Team:Paris Bettencourt/YonatanTest

From 2013.igem.org

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Revision as of 12:08, 25 October 2013

PROJECT
- OVERVIEW
- DETECT
- TARGET
- INFILTRATE
- SABOTAGE
ACHIEVEMENTS
HUMAN PRACTICE
SAFETY
COLLABORATION
- COLLABORATION
- SENSIGEM
NOTEBOOK
TEAM

@iGEM_Paris

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.

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.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

+33 1 44 41 25 22/25

team2013@igem-paris.org

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          <center> <img height="60%" src="https://static.igem.org/mediawiki/2013/9/99/PB_fig_target.png"/></center>
-         <p style="margin-top:-60px;font-size:14px">After expression of the Cas9 and gRNA, the gRNA guides the Cas9 to the target sequence, the kanamycin resistance. There, the Cas9 generates a double strand break. This activates the SOS response. The reporter LacZ is under the pRECA promoter, which gets activated during the SOS response and we get hence a blue cell, if the resistance gene has successfully been detected.</p>
+         <p style="margin-top:-60px;font-size:14px">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.
+</p>
        </div>
      </div>