Team:UCL/Tests

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.

	Registry ID	Name / Function	Source	Size
U	BBa_K314103	IPTG-inducible LacI Expression Cassette	Spring 2014 BioBrick Distribution. Plate 1, Well 4D.	1638 bp
T	BBa_J04450	RFP Coding Device	Spring 2014 BioBrick Distribution. Plate 4, Well 4B.	1069 bp
T	BBa_R0010	IPTG-inducible LacI Promoter	Spring 2014 BioBrick Distribution. Plate 3, Well 4G.	200 bp
T	BBa_B0034	Ribosomal Binding Site (RBS)	Spring 2014 BioBrick Distribution. Plate 4, Well 1N.	12 bp
T	BBa_K518012	RBS + RFP + double Terminator	Spring 2014 BioBrick Distribution. Plate 1, Well 18C.	828 bp
N	BBa_K206000	pBAD Strong Promoter	Spring 2014 BioBrick Distribution. Plate 3, Well 14A.	130 bp
! N	BBa_R0011	LacI-Regulated, Lambda pL Hybrid Promoter	Spring 2014 BioBrick Distribution. Plate 2, Well 6D.	55 bp
! N	BBa_B0012	Transcription Terminator for E. coli RNA Polymerase	Spring 2014 BioBrick Distribution. Plate 2, Well 2B.	41 bp

We plan to create a complete synthetic azo dye decolourising device in E. coli which incorporates several different independent enzymes that act on azo dyes and their breakdown products. After evaluating their individual breakdown characteristics, we aim to investigate the potential synergistic action of these enzymes in a single synthetic E. coli device and design a bioprocess which could be used to upscale the method to an industrial context.

In an industrial setting, these enzymes would work sequentially in a bioreactor with preset dynamic conditions. First, azoreductase will cleave the azo-bond (N=N), producing a series of highly toxic aromatic amines. Then, these compounds will be oxidised by lignin peroxidase, laccase and bacterial peroxidases, completing decolourisation and decreasing toxicity levels.

The complementary action of azoreductase, lignin peroxidase, laccase, and bacterial peroxidases will be studied in order to find out the best possible approach of sequential reaction, and this core degradation module will be extrapolated to other areas such as BioArt projects and work on algal-bacterial symbiosis.

This non-specific enzyme was isolated from Bacillus subtilis, although it is also found in other bacterial species. It starts the degradation of azo dyes by cleaving the azo bond.

The products of this cleavage varies greatly among different dyes, but are generally aromatic amines. This azo cleavage does not only occur with azo dyes, but also with other molecules like Sulfasalazine. We will isolate this enzyme from B. subtilis and convert it to BioBrick format via polymerase chain reaction (PCR).

Another azoreductase that we will be using is isolated from Pseudomonas aeruginosa. It functions in the same way as Azoreductase R1 - cleaving the azo bond - but it is intended to work complementary with it, in order to cover a wider spectrum of dyes more efficiently.

Like the previous azoreductase, this BioBrick will be constructed using PCR. A promoter and a ribosomal binding site (RBS) will then be added to create a functioning composite device.

Usually found in white-rot fungi species, its main function in nature is to participate in lignin-degrading processes by these organisms. However, it has also been found to play a role in azo dye degradation and decolourisation.

This enzyme, like laccase, would be incorporated in the second step of the reaction to oxidise the products of the azo bond cleavage, in order to achieve greater detoxification. The sequence for the enzyme will be ordered and synthesised, including the BioBrick prefix and suffix. Again, it will function together with a promoter and a RBS.

Found in B. subtilis, the physiological function of this newly discovered enzyme is still unclear, although it has shown effectiveness in degrading lignin and azo dyes, which makes it useful for us. It is not as effective as PpDyP for most compounds, but very efficient in degrading ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)).

The BioBrick will be constructed via PCR.

This enzyme is found in P. putida. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide variety of substrates very efficiently. This will broaden the spectrum of action of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters.

This BioBrick will be constructed via PCR.

This enzyme is found in P. putida. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide variety of substrates very efficiently. This will broaden the spectrum of action of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters.

This BioBrick will be constructed via PCR.

This enzyme is found in P. putida. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide variety of substrates very efficiently. This will broaden the spectrum of action of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters.

This BioBrick will be constructed via PCR.