Our literature search identified a number of bacterial species that exhibited an azo reducatase phenotype. We were able to obtain B. subtilis strains from ? and extracted the genomic from this using a Promega Wizard Genomic DNA extraction kit. We then ran a gel to determine whether we had successfully extracted the genomic DNA.

Experiment 1: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit

We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expresing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.

Experiment 2: Transforming E. coli with Azo-reductase plasmids

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Experiment 3: Diagnostic digest of azo-reductase plasmids

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Experiment 4: Creation of azo-reductase BioBrick parts from plasmids

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Experiment 5: Diagnostic digest of azo-reductase BioBrick parts

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Experiment 6: Assembling azo-reductase BioBrick Device(s)

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Experiment 7: Characterisation of azo-reductase BioBrick devices

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