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Revision as of 13:49, 22 October 2017

Lab

Composite Part

Facing back to the Etendard glacier, in between the peaks of Maurienne and the ones of Oisan.
Credits: Estelle Vincent

1. Generalities

BBa_K2299000 is a cholera detector part, its activation allows the detection of 39bp of CTX bacteriophage RstA gene.
Cholerae detector part
Biobrick nameBBa_K2299000
RFC CompatibilityRFC 10; RFC 12; RFC 23; RFC 25
Backbones pSB1C3
Submitted byiGEM Grenoble-Alpes 2017 team
We - iGEM Grenoble-Alpes 2017 team- focused on the creation of a system which would enable the detection of a short sequence present in Vibrio cholerae genome. This sequence is a fragment of bacteriophage CTX DNA, involved in Vibrio cholerae’s pathogenicity by integrating a part of its own genome which contains genes for cholera toxins production.
The target is a 39 bp sequence found in CTX RstA gene (implicated in the CTX phage DNA replication and integration), present in Vibrio cholerae O1 and O139. This chosen target is flanked with AluI restriction sites, so its digestion is necessary to free the target fragment with correct length.
The detector construction that we created - a probe that binds perfectly to the target - was inserted in psB1C3 containing BBa_J04450 allowing the emission of red fluorescence.
Finally, the goal is to detect fluorescence with SnapLab: it is our homemade detection kit, displaying the results of the detection by measuring the fluorescence intensity with a smartphone.

2. Design & functioning

The design of this detector has been inspired by Cork Ireland 2015 team : they created different detectors based on the perfect complementarity of the double strand DNA. Thus, we decided to create a similar detector for cholera’s pathogen.
BBa_K2299000 has been designed in two parts:
  • Bioinformatics work, aiming to find a target, build a probe depending on the target, and find an appropriate plasmid backbone to carry the probe.
  • Construction of the detector in practice, i.e. insertion of the probe inside the chosen backbone.
    • Then the probe is activated (via a quadruple digestion) allowing the opening of a “window” in BBa_K2299000, so the target can bind to the probe by perfect complementarity. Plasmid is re-circularized and can be transformed.
    Then the red fluorescence emitted by the bacteria that incorporated the plasmid with the target is detected thanks to a smartphone camera fixed on our detection kit, SnapLab, inside which activation and binding happened.
    Figure 4 : Schematic overview of the construction of the DNA detector
    All the protocols can be found HERE, please have a look !

    Designed by iGEM Grenoble-Alpes 2017 team | Site Map