Part:BBa_K1336007:Experience

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LEC + BsDyP

After having studied the viability of BBa_K1336003, 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 measuring bacterial OD at regular intervals of 1 hour, in the different media. Each of the tubes from which the samples were extracted contained initially 10mL of LB medium (formulated by mixing 25 gr of Sigma-Aldrich L3522 Luria broth with 1 L of Milli-Q water). Apart from the plasmid-free controls, each tube also contained 10uL of 25ng/uL Chlorampehicol. The cells used were Invitrogen™ DH5α™, which show the following genotype: F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1. All cultures were grown overnight at 37 ºC and shaking at 250rpm, in order to allow them to reach stationary phase, to then inoculate the dyes and measure the effects. All the time frames specified below have as a starting point the inoculation of the dyes, NOT the cells.

To these tubes, 100uL of the three different concentrations for each of the two dyes were added, to give the desired final concentrations as specified below. To the controls, 100uL of sterile water was added. They were then incubated for the time frames indicated in the figures below, and at the specified time points two samples of 200uL were taken into two cuvettes to then be diluted into 1.8mL of LB (from the same batch as that found in the culture tubes). The absorbance shown on the graphs is the absolute value, not the dilution. Readings were taken in a standard spectrophotometer at 680nm; 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 for this assay can be found here.

The first 24 hours of incubation at 37ºC shaking showed subtle decolourisation. This is represented in the graphs below. RB5 at its highest concentration (0.5 mg/mL) seems to present the most significant drop in absorbance 6 hours of inoculation of the dyes. The decolourisation obtained is not as high as expected, possibly due to non ideal temperature for the activity of this particular enzyme.

Figure 1a - BBa_K1336007 LEC+BsDyP Azo-degradation device shows indicative effectiveness on Reactive Black 5 after 24 hours of shaking incubation. Graph showing the indicative decolourisation activity of E. coli DH5Gα transformed with BBa_K1336007 on RB5,
at three different concentrations in LB media. Cells were incubated for 24 hours at 37 ºC and shaking at 250rpm. Please note that OD measurements are considerably higher in dye-contaminated waters due to the absorbance of the azo-dye. OD measured at 600nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

Figure 1b - BBa_K1336007 LEC+BsDyP Azo-degradation device shows indicative effectiveness on Acid Orange 7 after 24 hours of shaking incubation. Graph showing the indicative decolourisation activity of E. coli DH5Gα transformed with BBa_K1336007 on AO7, at three different concentrations in LB media. Cells were incubated for 24 hours at 37 ºC and shaking at 250rpm. Please note that OD measurements are considerably higher in dye-contaminated waters due to the absorbance of the azo-dye. OD measured at 480nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

The second step of the decolourisation assay, however, showed a much more dramatic decolourising effect after 30 hours. After centrifugation, it could be observed that the supernatant for the AO7 (0.0155 mg/mL) with cells that contained the plasmid had a less intense colour than the plasmid-free control, where no degradation was expected.


Figure 2 - BBa_K1336007 Decolourisation of 0.0155 mg/mL Acid Orange 7 by BBa_K1336007 after 30 hours stationary at room temperature. Samples were centrifuged in order to measure the OD values of the supernatants. On the right, the supernatant of the plasmid-free cells. On the left, the supernatant of the sample with BBa_K1336007-containing cells, showing a less intense colour than the control. Picture taken after 24 hours of incubation at 37 ºC and 250rpm plus 30 hours stationary at room temperature.

This decolourisation was confirmed by spectrophotometric analysis of the samples, as shown in the figures below.

Figure 3a - BBa_K1336007 BsDyP Azo-degradation module is capable of degrading Acid Orange 7 (AO7) dye-contaminated waste waters at room temperature. 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 3b - 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.

These assays show the effectiveness of the BBa_K1336007 degradation device in decolourisation of the two tested dyes at the indicated conditions. It remains unclear why most of the decolourisation took place in the second part of the assay; a possible explanation fot this is that the temperature post-incubation was more optimal for BsDyP function.

The conclusion drawn for the decolourisation experiments is that it would be possible to integrate the dye-decolourising device BBa_K1336007 in a bioprocessing context aimed towards the biological degradation of azo-dye contaminated waters, as it seems to be effective in partially degrading sulphonate azo-dyes Reactive Black 5 and Acid Orange 7.

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