Team:Paris Bettencourt/Results

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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.
  • Confirmation of site-specific binding and DNA double-strand breaks generated by the gRNA-Cas9 complex in the kanamycin resistance casette.
  • Testing the new assembly standard for our cloning.

CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli. We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, blue bars). The strains were co-transformed with two plasmids. One with the Cas9 construct, the other with the anti-Kanamycin gRNA. WT E.coli could be efficiently transformed with one or both plasmids, as determined by selective plating. However, KanR E. coli could not be efficiently transformed with both. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette, with about 99% efficiency.

Target

Background

SirA is an essential gene in latent tuberculosis infections.

Aims

To perform a 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 analysis.

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.

TDMH 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.

Sabotage

Background

One of the main concerns about tuberculosis today is the emergence of antibiotic resistant strains.

Aims

Our objective is to make an antibiotic-resistant bacterial population sensitive again to the selfsame antibiotics.

Results

  • Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins.
  • Showed theoretically that fitness cost is critical for the stable propagation of a genetic element in a population.
  • Successful conversion of antibiotic resistant population of E. coli to a sensitive state (on two different antibiotic resistances).

Our synthetic phage conveys antibiotic-sensitivity to an antbiotic-resistant population. The anti-Chloramphenicol phage system effectively killed 99.1% of the Chloramphenicol resistant population and the anti- Kanamycine phage effictevely killed 99,5% of the Kanamycine resistant population.

Modelling

Population Dynamics Model

This model investigates the effects of the fitness-cost of a genetic element on it's spread in a bacterial population, based on a phagemid helper system.

Structural Analysis of SirA

Using Swiss pdb we demonstrated the superimposed 3D structures of Mycobacterium tuberculosis SirA and Escherichia coli CysI highlighting their similarities and differences. Both proteins are important in their respective sulphite reduction pathways. We then predicted the effect of a small drug target based on SirA's structure.

Flux Balance Analysis

We used an E. coli model iJR904 obtained from BiGG database as a starting model and obtained a growth rate represented by the f value of 0.9129. We then deleted the reaction ‘SULR’ which encodes for the sulphite reduction pathway involving cysI and obtained a f value of -8.63596783409936e-13 indicating that the sulphite reduction pathway is required for growth.

Human Practice

Technology Transfer

An essay that addresses the issue of designing a technology aimed at "developing" countries, rather than at “developed” ones: a typical case of technology transfer.

TB in France

Analysis of the social, medical and political aspects of the management of tuberculosis in France. Synthetic biology may help in many ways such as treatments, drug development, diagnostic. We also give advice on how to introduce it in clinics.

Gender Study

A comprehensive and quantitative study of gender in(equality) in iGEM and synthetic biology. A database was gathered and statistically analysed in order to depict sex ratio of iGEM teams' students and supervisors.


Gender balance and success in iGEM
Winning teams have a significantly higher number of women and are more Gender balanced.

Collaboration

SensiGEM

SensiGEM is the iGEM Biosensor database generated by the teams Paris Bettencourt 2013 and Calgary 2013. In this database you can find fast and easy what biosensor projects were already done by past iGEM Teams. To be able to select the projects that fit into the database, we also collaborated to compose a joint definition a biosensor.

BGU iGEM Team from Israel

A mutual part characterization. We characterize the promoter units produced by the lac/ara-1 promoter of cI, a repressor of their constructed kill switch. In return, BGU characterizes our TDMH biobrick protein expression levels by Western Blot.