Part:BBa_K1336003:Experience

Bacillus subtilis dye-decolourising peroxidase

After having confirmed that Reactive Black 5 and Acid Orange 7 are not toxic and have no effect on E. coli DH5α growth 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 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 at 37 ºC and shaking at 250rpm.

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.



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 2a - 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 2b - 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.

A functional device for this part using a Lac-induced promoter was characterised here

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References:
1.-Santos A, Mendes S, Brissos V, Martins LO (2014)New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications. Appl Microbiol Biotechnol (2014)98:2053-2065