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Team:Paris Bettencourt/YonatanTest
From 2013.igem.org
Detect
Background
CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.
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.
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.
Aims
Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.
Target
Background
SirA is an essential gene in latent tuberculosis infections
Results
- Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media
- Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis
- Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis
Aims
To perform an drug screen targeted at the sirA gene from mycobacteria
Infiltrate
Background
Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics.
Results
- We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in E.coli and showed that it is highly toxic to mycobacteria in culture.
- We expressed the lysteriolyin O (LLO) gene in E. coli and showed that it is capable of entering the macrophage cytosol.
- We co-infected macrophages with both mycobacteria and our engineered E. coli to characterize the resulting phagocytosis and killing.
BioBricks
Aim
To create an E. coli strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.
Sabotage
Background
One of the main concern about tuberculosis today is the emergence of antibiotic resistant strain
Results
- Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins
- successful conversion of antibiotic resistant population of E. coli to a sensitive state
BioBricks
Aims
Our objective is to make an antibiotic-resistant bacterial population sensitive again to those same antibiotics.
+33 1 44 41 25 22/25
team2013@igem-paris.org 
