Synthetic Gene Pathway

ATC induces the circuit.

TetR is disabled by ATC.

The disabled TetR can no longer inhibit the transcription of the pathogen imitating sRNA .

The sRNA can bind to the constitutively expressed riboregulated gRNA, exposing the targeting domain.

The exposed dCAS9 recognising cis-acting factor within the gRNA binds to the constitutively expressed dCAS9 protein.

Once enabled the dCAS9 protein will bind to the LacIq promoter, inhibiting the expression of the LacI repressor protein.

Once the LacI repressor is inhibited, every pLAC will be upregulated due to lack of inhibition

The sfGFP will be synthesised from the pLAC, resulting in a green fluorescence.

The sRNA and dCAS9 are found on a Kanamycin resistant plasmid.

The gRNA and GFP are found on a Chloramphenicol plasmid.

The LacI is found on the Z1 cassette found in MG1655 Z1 cells. The Z1 cassette contains a set of repressors for the cell to utilise. Initially we designed our circuit to respond to the EndA gene in different strains of E.coli. Our project was going to be used to determine whether an unidentified strain of lab E.coli was MG1655 or DH5 alpha - or more importantly, whether or not the bacteria had a specific EndA mutant which could affect miniprep results. After our interview with Professor David Coleman we changed the course of the project and our entire iGEM team to focus on Legionella detection and water safety as a whole. We integrated his design suggestions into our plasmid sequence design and function. The new plasmids were designed to target Legionella.

Team:Warwick/Design

Design

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igem@warwick.ac.uk

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