1. Enzyme 01: AzoR, Azoreductase
2. Enzyme 02: 1B6, Azoreductase heat-stable mutant
3. Enzyme 03: CotA
4. Enzyme 04: BsDyP, Bacilus subtilis dye-decolourising peroxidase
5. Enzyme 05: PpDyP, Pseudomonas putida dye-decolourising peroxidase
6. Enzyme 06: LiP, Lignin peroxidase / Ligninase

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Extraction of Bacillus subtilis genomic DNA

Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including B. subtilis and P. aeruginosa. We were able to obtain a B. subtilis strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the B. subtilis genomic DNA.

Transforming E. coli with Azo-reductase plasmids

We were gratefully provided with a set of five plasmids from a group of researchers working at the University of Lisbon, Portugal who are researching how azo-dye degrading enzymes function and who were keen to collaborate with us. These plasmids contained a number of genes encoding azo-dye degrading enzymes from both B. subtilis and P. putida including mutated forms found to exhibit enhanced degradation 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 NEB5alpha competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.

Name	Function	Source	Concentration	Sequence	Initial Plasmid / Vector	Comments
pAzoR	FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 48 ng/uL,	597 bp [Check! Not 612 bp?]	Expression vector pET-21a (+) (ampicillin resistant (ampR)), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon
p1B6 (AzoR 1B6)	Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 68 ng/uL,	597 bp [Check! Not 612 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.
pCotA	Spore Coat Protein Laccase	Bacillus subtilis	Miniprep, 103 ng/uL	1733 bp [Check! Not 1539 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NheI and BamHI.	Plasmid provided by Lisbon.
pBsDyP	Dye Decolourising Peroxidase BSU38260	Bacillus subtilis	Miniprep, 51 ng/uL,	1251 bp	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.
pPpDyP	Dye Decolourising Peroxidase PP_3248	Pseudomonas putida	Miniprep, 55 ng/uL	861 bp [Check! Not 864 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.

Diagnostic digest of azo-reductase plasmids

After successfully transforming these plasmids into competent E. coli NEB5alpha cells we then performed a diagnostic digest and gel electrophoresis experiment to ascertain that these plasmids contained the gene we expected. Each plasmid was digested using two restriction enzymes chosen to digest DNA as specific points on the plasmids and create fragments of known length which we could then confirm using gel electrophoresis.

Creation of azo-reductase BioBrick parts from plasmids

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Diagnostic digest of azo-reductase BioBrick parts

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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.

Assembling azo-reductase BioBrick Device(s)

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Characterisation of azo-reductase BioBrick devices

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