Experiments

Experiment 1: Extraction of Bacilius subtilis genomic DNA

Our literature search identified a number of bacterial species that were found to degrade azo dye compounds including B. subtilis and P. aeruginosa. We were able to obtain a B. subtilis strain from ? and extracted the genomic from this using a Promega Wizard Genomic DNA extraction kit so that we could amplify the azo-reducatase gene (AzoR1). After completing the genomic DNA extracton we produced a gel to determine whether we had successfully extracted the genomic DNA.

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

We were gratefully provided with a set of plasmids from a group of researchers at the University of Lisbon, Portugal who were also working to understand how azo-reductase enzymes function. These plasmids contained a number of azo-reductase genes from both B. subtilis and P. aeruginosa including mutated forms found to exhibit enhanced reductase activity. As the DNA concentration of the plasmids we were sent was insufficient to perform PCR amplification on we transformed each of these plasmids into our E. coli NEB5lpha competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.

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 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 6: Assembling azo-reductase BioBrick Device(s)

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Experiment 7: Characterisation of azo-reductase BioBrick devices

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