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Team:UCL/Science/Results/Deg
From 2014.igem.org
Degradation
Cloning of Lignin Peroxidase - BBa_K500000
Lignin peroxidase is known to have dye-decolorising activity, being particularly effective when the substrates are sulphonated azo-dyes. This led us to investigate whether we could incorporate the Lignin Peroxidase (BBa_K500000) BioBrick submitted by Tianjin iGEM 2010 into our project and provide further characterisation; there was no available data in the Registry regarding to the functionality or viability of this fungal part in E. coli.
The sequence for this part, found here (insert link), was codon-optimized for E. coli K12 and synthesis was requested along with the BioBrick prefix and suffix. 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 issues of (BBa_K500000) in E.coli.
The gene synthesis was carried out by GeneOracle, who provided us with data for the toxicity issues that they encountered. In summary, in-vitro synthesis presented no particular problems, but when the sequence was cloned into the destination strain, the sequence was modified with a number of mutations, deletions, and modifications, that made it impossible to recover the original gene. Cloning was attempted on a pGOv4 plasmid; detailed information about this plasmid can be found in the files below.
pGOv4 Diagram
pGOv4 Information
Figure 1 - 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.
The toxicity issues that arise when trying to express this fungal enzyme in E. coli can explain why no previous attempt to characterise the part recorded in the registry have been successful. Further characterisation of this toxicity, as well as attempting to troubleshoot it, could involve the subcloning of the part into a low-expression vector.
The inability to characterise the decolourising abilities of Lignin Peroxidase made us look towards a bacterial alternative; Bacillus subtilis dye-decolorising peroxidase. This enzyme, described in literature as the bacterial equivalent of lignin peroxidase due to the similarity of their mechanisms was cloned successfully, and the data regarding its toxicity and ability to decolorise azo-dyes is developed below.
Characterisation of BsDyP
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 for this assay can be found here
Figure 2 - 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 3a - 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.
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 3b - 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 compatible with successful DH5α 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 absorptions for each dye. The full protocol 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 4a - 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.
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 4b - 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 5 - 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 6a - 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.
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 6b - 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.
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.
