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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.
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) and a kanamycin resistant strain (KanR, red) were co-transformed with a plasmid carrying the Cas9 construct, and a second plasmid carrying the anti-Kanamycin gRNA. WT was successfully transformed with one or 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.
Target
Background
SirA is an essential gene in latent tuberculosis infections
Aims
To perform an drug screen targeted at the sirA gene from mycobacteria
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
MycoSIR E. coli depend on our synthetic pathway for growth. E. coli strain BL21(DE3) was deleted for cysI and transformed with the three genes of the mycoSIR pathway expressed from IPTG-inducible T7 promoters (red). Wild-type (blue), uninduced (purple) and pathway-minus (gold) strains were used as controls. Both time course growth curves (A) and final ODs (B) reveal that the complete, induced pathway is required for growth
Infiltrate
Background
Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics.
Aims
To create an E. coli strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.
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.
FTDMH expression kills mycobacteria in culture. We mixed E. coli and WT M.smegmatis in LB media. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.
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