Biobricks

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.

Vector	iGEM Registry Part Code	BioBrick	Function
	BBa_K1336000	Azoreductase R2	This part encodes an enzyme for cleaving the N=N bond in azo dyes, identified from Bacillus subtilis ...
	BBa_K1336001	Azoreductase 1B6	This part also cleaves the N=N bond in azo dyes, but was isolated from Pseudomonas aeruginosa ...
	BBa_K1336003	Lignin Peroxidase	This part, found in white-rot fungi, participates in lignin-degrading processes, which also plays a role in azo dye degradation and decolourisation ...
	BBa_K1336004	Bacillus subtilis dye-decolorizing peroxidase (BsDyP)	This part is a newly discovered enzyme that appears to be effective in degrading lignin and azo dyes, however, its physiological functions have not yet been fully characterised ...
	BBa_K1336005	Pseudomonas putida MET94 dye-decolorizing peroxidase (PpDyP)	This part is another relatively novel enzyme that has not yet been studied in detail, but seems to be able to effectively oxidise many substrates including: azo dyes, anthraquinones, phenolic compounds, manganese, and veratryl alcohol ...

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) by a double reduction using NADPH as a cofactor, 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, to the point that the final products of the process are less toxic than the intact dyes themselves. 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, trying to set up the foundations for a synthetic ecology.

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Detailed BioBrick functions

Azoreductase R2 (BBa_K1336000)

This non-specific enzyme was isolated from Bacillus subtilis, although it is also found in other bacterial species, including those inhabiting the human intestine. It starts the degradation of azo dyes by cleaving the azo bond, composed of two nitrogens linked by a double bond (N=N), which is characteristic of all azo dyes. 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, a drug that is broken down in the gut to release compounds that fight bowel disease and arthritis. We will isolate this enzyme from B. subtilis and convert it to BioBrick format via polymerase chain reaction (PCR).

Azoreductase 1B6 (BBa_K1336001)

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.

Lignin Peroxidase (BBa_K1336003)

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, by oxidative processes. 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.

Bacillus subtilis dye-decolorizing peroxidase (BsDyP) (BBa_K1336004)

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.

Pseudomonas putida MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336005)

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, like azo dyes, anthraquinones, phenolic compounds, manganese or veratryl alcohol. This will broaden the spectrum of action of our decolourising device, going further just azo dyes, and thus being able to degrade other toxic compounds typically found in industrial wastewaters. BioBrick will be constructed via PCR.