Difference between revisions of "Part:BBa K2684000"

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CotA Laccase of B.subtilis

CotA Laccase is an endospore type protein secreted from B.subtilis Part BBa_K2684000 was improved from previous part BBa_K1336002: https://parts.igem.org/Part:BBa_K1336002. A point mutation has been made to eliminate the EcoRI cutting site so that we can meet the standard of RFC10

Sequence and Features

Usage and Biology

CotA is a polyphenol oxidase which has capability to decolorize a wide range of dyes, as the catalyst. It has been reported that CotA laccase decomposes dye efficiently even in harsh condition with 3.5% salinity and pH 11.6, which makes it a great catalyzer for dye decomposition.

The CotA gene was using PCR from B. subtilis WT 168 genome, and expressed it in E. coli BL21. We quantified the activity of recombinant CotA using a commercial assay kit in which the CotA catalyzes the oxidation of ABTS. When oxidized by laccase, ABTS would turn to a bullish-green color that has an absorbance peak at 420nm.

The enzyme activity was measured and calculated based on the weight of our sample. Formula: laccase activity (nmol/min/g) = ΔA /ε(ABTS mmolar extinction coefficient) / d * V (total volume of reaction) / V (volume of sample in the reaction, 0.025mL) / W (sample mass, g) * V (extracted liquid added, 1mL) / T (reaction time, 3 minutes) = 130 *ΔA / W.

For more experimental details, please visit http://2018.igem.org/Team:SHSBNU_China/Experiments

References

Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.

“Help:Standards/Assembly/RFC10.” Help:Standards/Assembly/RFC10, parts.igem.org/Help:Standards/Assembly/RFC10.

The followed experience is from 2019 iGEM team Worldshaper-XSHS

As we known from the literatures, CotA laccase (BBa_K2684000) is a polyphenol oxidase which has capablity to decolorize a wide range of dyes. However, from the previous study, we noticed that this Biobrick had been characterized the performance by Enzyme activity but none of the degradation data. So this summer, our team recharacterized decolorization ability of the biobrick BBa_K2684000 (coding for CotA) using one of azo dyes called reactive red x (“RR” for short). Details are as followed.

SDS-Page Verification

SDS-PAGE experiments were performed to verify the expression of the enzyme protein. CotA-, CotA+ represent no induction and after induction by IPTG, respectivley. By reviewing the literature, we found that CotA has a protein size 59.9 kDa. After comparison with the images, we found that all proteins were successfully expressed under the induction of IPTG.

Decolorization Ability Test

We expressed CotA in E. coli BL21 and divided the cells into two groups: one group was induced by IPTG, which was labeled as CotA +, while the other group without IPTG was labeled as CotA -. We detect the absorbance at regular intervals, the results was showed in Figure 1.

Figure 1. Absorbance change of RR catalyzed by cotA

As shown in Figure 1, there were not significant differences between the IPTG induced group and the control group until the treatment time exceed 12 hours. And the absorbance value decreased significantly, which proved that the degradation effect was significant after IPTG induced expression. After 48 hours, the absorbance value has been reduced to 0.7, which is better than that of the control group, indicating that cotA has a better decolorization effect under the induction of IPTG. At the same time of the absorbance detection of the supernatant, we also retained the bacteria and observed them. It can be seen from Figure 2 that in IPTG induction group, the color of bacteria is purple, which is closer to the color of dye. However, in the control group without IPTG, the color of bacteria was yellow, which was more similar to the color of the mixture of bacteria and pigment, so it was speculated that its adsorption on dye was very weak. According to the color observation of the supernatant in Figure 3, after 48h, the dye has been degraded to a large extent, and its color is obviously different from the original color.

Figure 2. Cell color change under cotA catalysis

Figure 3. Color change of supernatant under the catalysis of cotA

Actual sewage test

In additon, we also do a actual sewage test. The wastewater from the printing and dyeing factory was added with the same concentration of dye RR as the previous experiment for RR degradation test. In order to ensure the consistency of the experimental method, we have done the dye degradation test of the sewage group and the experimental group at the same time, and recorded the light absorption value of the dye in 24 hours, 48 hours and 72 hours. The results are as follows: In the experimental group, we repeated the experiment according to the method of functional test. In this result, cotA showed good degradation effect on RR. It can be seen that the dye had been degraded by naked eyes at 48 hours.

In the sewage group, CotA can work normally, but there are also unexpected findings: in the sewage, the degradation effect of the enzyme is faster. At the same time, the control group which was not induced by IPTG also had obvious decolorization phenomenon. In view of this situation, we suspect that the reason may be the presence of degradation substances in sewage.

In order to verify the specific cause of this phenomenon, we did the following negative control experiments: adding wastewater treatment solution WR to LB culture medium directly, and taking dye RR in LB culture medium + pure water as the control. The results are as follows:

It can be seen that the sewage group without bacteria has a certain degradation effect. However, we have also observed mixed bacteria in the culture medium, but the possible degradation factors of the sewage itself can not be determined whether it is bacteria or other unknown substances, and further experiments are still needed to determine.