Team:UCL/Science/Experiment

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 expressing 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.

Searching through the literature, we identified a number of bacterial species (including Bacillus subtilis and Pseudomonas sp.)that have proven to degrade azo dye compounds [1][2][3][4].
We contacted the Microbial & Enzyme Technology Lab led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both B. subtilis and P. putida (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below).

In the meantime, Helina (in our team), was able to obtain B. subtilis and P. aeruginosa strains for us to test whether we could retrieve azo dye degrading enzymes from their genomes, specifically, the azo-reductase gene (AzoR). This would be the first step for our first azoreductase BioBrick.
We extracted the genomic DNA from B. subtilis strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azoreducatase gene (AzoR1) and create our first azoreductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the B. subtilis genomic DNA.

We were gratefully provided with a set of five plasmids from the Microbial & Enzyme Technology Lab led by Dr Lígia O. Martins at the Universidade Nova de Lisboa. They are currently researching how azo-dye degrading enzymes function and are 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 derivative competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.

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.

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.

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	Registry ID	Gene ID	Name / Function	Source	Size	Status
	BBa_K1336000	AzoR	FMN-dependent NADH-azoreductase 1	Pseudomonas putida	612 bp	[In Progress]: primers designed
	BBa_K1336001	1B6	AzoR heat-stable mutant	Pseudomonas putida	612 bp	[In Progress]: to remove 2 illegal PstI sites
	BBa_K1336002	CotA	Spore Coat Protein Laccase	Bacillus subtilis	1542 bp	[In Progress]: primers designed
	BBa_K1336003	BsDyP	Dye Decolourising Peroxidase BSU38260	Bacillus subtilis	1251 bp	[New BioBrick Part]: submitted
	BBa_K1336004	PpDyP	Dye Decolourising Peroxidase PP_3248	Pseudomonas putida	864 bp	[In Progress]: primers designed
	BBa_K1336005	ispB RNAi	RNAi of Octaprenyl Diphosphate Synthase fragment	Escherichia coli, K12 strain	562 bp	[New BioBrick Part]: submitted
	BBa_K1336006	LacIEC+ispB	IPTG inducible ispB RNAi	Escherichia coli, K12 strain	2208 bp	[New BioBrick Device]: submitted
	BBa_K1336007	LacIEC+BsDyP	IPTG inducible BsDyP	Bacillus subtilis	2895 bp	[New BioBrick Device]: submitted
	BBa_K729006	CueO	Laccase	Escherichia coli	1612 bp	[In Progress]: ascertaining identity
()	BBa_K500000	LiP	Lignin Peroxidase	Phanerochaete chrysosporium	1116 bp	[Improved Characterisation]: toxicity issues in gene synthesis.  [In Progress]: to subclone into pSB1C3/pSB3C5.
	BBa_K729004	nucB	Extracellular nuclease	Staphylococcus aureus	561 bp	[Improved Function]

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