Goodbye Azodye UCL iGEM 2014


Our BioBricks & how they lead to azo degradation

We have strove to complete a synthetic azo dye decolourising device in E. coli which incorporates several different independent enzymes that act on azo dyes and their breakdown products to create less toxic chemicals. 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.

Ideally, we wanted a Azo-Remediation Chassis (ARC), our BioBrick System, to be assembled as follows:

A further development on this prototype would be to have Bba_K1336000, the AzoR gene, to be inducibly transcribed by one promoter (say BBa_K314103, the LacI Expression Cassette) such that it is expressed in the reductive step of our azo dye remediation process. This would form a distinct BioBrick Device of "promotor A + AzoR + double terminator".
A secondary BioBrick Device (of "promoter B + gene 2 + double terminator") would follow this, where gene 2 would be one of our enzymes that function in the oxidative step of the azo dye remediation process, e.g. laccase or one of the dye decolourising peroxidases. A further tertiary BioBrick Device, with another oxidative enzyme would also be ideal. At least 2 oxidative enzymes are proposed as the enzymes are specialised for different substrates (as described in our BioBrick page.

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.

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. To ensure that the process is entirely bio-safe we have designed a xeno-biological modules that ensures that beyond the bioreactor our organism could not survive.

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]

Azoreductase (BBa_K1336000)

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

This azo cleavage, does not only occur with azo dyes, but also with other molecules like Sulfasalazine. We isolated this enzyme from B. subtilis and converted it to BioBrick format via polymerase chain reaction (PCR). However the site directed mutagenesis did not successfully remove the illegal restriction sites and therefore we could not characterise the effects.

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.

AzoR creates two or more aromatic amines (dependent on the number of azo bonds in the molecule) which are carcinogens. This is therefore only the first step of degradation. Some useful aromatic amines will be filtered off and sold as feedstock products for the fragrance industry which is further explained in the bioprocessing pages.

Spore Coat Protein Laccase (BBa_K1336002)

The laccase enzyme is a very non-specific oxidising enzyme. We intend to use it in concert with and as a second step to the azo reductase action to create no toxic products. Laccase will:

  • Break down azo bonds in specific azo dyes to non-toxic compounds
  • Polymerise non-specific azoreductase breakdown products to filterable, non toxic compounds
  • Oxidise specific azoreductase breakdown products to non-toxic compounds.

The spanning range of reactions that take place in laccase are largely due to the unspecific copper mediated oxidising active site. Known reaction products are below, however these are known to break down further in ways not yet tested via NMR.

Despite laccase’s unspecific active site, it cannot break down sulphonated dyes and hence oxidation of those must be left for the peroxidases (see next tab.)

Lignin Peroxidase (BBa_K500000)

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.

Bacillus subtilis dye-decolorizing peroxidase (BsDyP) (BBa_K1336003)

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_K1336004)

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.

Octaprenyl Diphosphate Synthase (ispB) (BBa_K1336005)

Octaprenyl diphosphate synthase is a crucial enzyme in the E. coli metabolism, being responsible for the synthesis of the side chains of isoprene quinones.

In order to knock down this gene, an anti-sense RNA sequence was designed in BioBick format via PCR. This would allow us to, in the future, develop through bio-directed evolution an E. coli strain completely dependent on certain synthetic compounds, such as azo-dyes.

Extracellular Nuclease (nucB) (BBa_K729004)

This part, submitted by the 2012 UCL iGEM team, was tested in the presence of azo-dye contaminants as a safety approach involving the degradation of extracellular DNA, and thus inhibiting DNA transfer between our synthetic organism and other bacterial species present in the environment.

More information on the characterisation of this part can be found in the results page.

SpyGFP (BBa_K239009)

This part, developed by the 2012 UCL iGEM team, was designed to function as a stress-sensor, producing GFP when exposed to shear stress.

We aimed to re-purpose this sensor, testing whether the presence of the azo-dyes in the medium was detected by the cells as "stress", with a subsequent production of GFP.

Contact Us

University College London
Gower Street - London
Biochemical Engineering Department
Phone: +44 (0)20 7679 2000

Follow Us