Team:UCL/Science/Results/Deg

Degradation

After having confirmed that Reactive Black 5 and Acid Orange 7 are not toxic and have no effect of ''E. coli'' DH5α in a wide range of concentrations, it was determined whether the dye-decolorizing BioBrick BsDyP BBa_K1336003 affected E. coli growth performance, both in standard LB medium and in media contaminated with RB5 and AO7 sulphonated azo-dyes.

This was carried out by measuring bacterial OD at 680nm at regular intervals of 1 hour, in the different media. The choice of wavelength aims to reduce to a minimum the interference caused by the strong absorption of the dyes, while still measuring bacterial density. Although high-concentration RB5 still shows an absorption much higher than the other samples, the curve is preserved and so it allows to analyse how the presence of dyes might interfere with bacterial growth. The full protocol fot this assay can be found here (insert link).



Figure 1 - BBa_K1336003 BsDyP Azo-degradation module preserves growth performance of E.coli DH5α in LB media. Graph showing that E.coli transformed with the BBa_K1336003 BsDyP Azo-degradation module shows comparable growth the plasmid-free control in LB media. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.



Figure 2 - BBa_K1336003 BsDyP Azo-degradation module is compatible with Acid Orange 7 (AO7) dye-contaminated waste waters. Graph showing that E.coli transformed with the BBa_K1336003 BsDyP Azo-degradation module is able to grow in LB media contaminated with AO7 dye. Please note that OD measurements are considerably higher in dye-contaminated waters due to the absorbance of the azo-dye. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.



Figure 3 - BBa_K1336003 BsDyP Azo-degradation module is compatible with Reactive Black 5 (RB5) dye-contaminated waste waters. Graph showing that E.coli transformed with the BBa_K1336003 BsDyP Azo-degradation module is able to grow in LB media contaminated with RB5 dye. Please note that OD measurements are considerably higher in dye-contaminated waters due to the absorbance of the azo-dye. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.


These assays confirm that the presence of the plasmid containing the BsDyP sequence has no detrimental effect on DH5α growth, as it is always comparable to or higher than the plasmid-free control. This means that the BsDyP BioBrick BBa_K1336003 would be fully compatibe with successful DH5alpha growth in industrial, highly azo-dye contaminated environments.

The next step was to investigate the functionality of the Azo-degradation device BBa_K1336007, composed of BsDyP BBa_K1336003 plus the IPTG-inducble BBa_K314103 expression cassette, in decolourising several Azo-dye contaminated waste-waters. This was carried out by growing the BBa_K1336007 containing cells over-night, allowing them to reach stationary phase while already expressing the part, to then inoculate the dyes at the different concentrations. The differences in absorbance between plasmid-containing samples and plasmid-free was measured by OD at the corresponding maximum absorptiions for each dye. The full protocol can be found here (insert link).


Figure 4 - BBa_K1336007 BsDyP Azo-degradation module is capable of degrading Acid Orange 7 (AO7) dye-contaminated waste waters. Graph shows that in comparison to the plasmid free control, E.coli transformed with the BBa_K1336007 BsDyP Azo-degradation device is able to decolourise AO7 (0.155 mg/mL) dye contaminated LB media after being induced by 1mM IPTG. Inoculations were grown at 37 degrees and 250rpm for 24 hours and then left stationary for a further 30 hours at room temperature. The samples were centrifuged, and OD480nm measurements were taken of the supernatant at the end of the 54 hour experiment. Error bars indicate SEM, n=2.



Figure 5 - BBa_K1336007 BsDyP Azo-degradation module is capable of degrading Reactive Black 5 (RB5) dye-contaminated waste waters. Graph showing that in comparison to the plasmid free control, E.coli transformed with the BBa_K1336007 BsDyP Azo-degradation device is able to decolourise RB5 (0.5 mg/mL) dye-contaminated LB media after being induced by 1mM IPTG. Inoculations were grown at 37 degrees and 250rpm for 24 hours and then left stationary for a further 30 hours at room temperature. The samples were centrifuged, and OD600nm measurements were taken of the supernatant at the end of the 54 hour experiment. Error bars indicate SEM, n=2.


Lignin Peroxidase - BBa_K500000
In order to widen our Azo-degrading and de-colourising toolbox, we also set out to investigate whether we could incorporate the Lignin Peroxidase (BBa_K500000) BioBrick submitted by Tianjin iGEM 2010 into our project and provide further characterisation. While we were able to get this fragment synthesised and sent to us, the toxicity of the DNA fragment in E.coli prevented us from obtaining any decolourisation data. However, we were still able to further characterise the experimental use of (BBa_K500000) in E.coli.



Figure 6 - Lignin Peroxidase BBa_K500000 BioBrick is toxic in E.coli DH5α. Graph showing that while it was possible to confirm the successful synthesis of BBa_K500000 in vitro, it was not possible to carry out any in vivo transformations of the DNA fragment into E.coli DH5α.

Conclusions: DANIEL TO FILL OUT