Part:BBa_K2684000
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1348
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 286
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.
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.
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.
The followed experience is from 2021 iGEM team THIS-China
THIS-China also explored the degradation ability of the protein CotA. We obtained slightly different data than the previous group, even though we have used the same RR red color as them. The data are shown below. CotA+ represents CotA mixed with IPTG (got activated and expressed), and CotA means that there is no IPTG added. From this data, we can see that the color degradation is already clear during the first 24h, and in the next 24h, the absorbance barely decreases for CotA+. Therefore, we came to the conclusion that the color changing time is around 24h.
The followed experience is from 2021 iGEM team KEYSTONE
KEYSTONE had explored the lactase function as an autocrine function and can also act as a fusion chaperone to assist in the secretion of the target protein. In addition, laccase is a good indicator when it acts as a fusion partner, and it can react with ABTS when it is secreted into solid medium.
We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Figure 4A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Figure 4B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity; however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved, showed by Figure 4. As shown by figure 4, CotA-Lcp-HlyA has achieved successful secretion.
The followed experience is from 2021 iGEM team Whittle
Whittle had expressed laccase CotA in a pET28a-T7 promoter system(A)，and purified the laccase protein utlized His-tag purification reagent. We designed a 6x his-tag at the C terminal of CotA sequence and insert this part into pET28a expression Vector. Then we transformed the plasmid into E.coli BL21(DE3) strain and induced the protein expression under 0.3mM IPTG and 20C degree. The sds-page result showed we successfully purified the target protein with the size of 60kDa(B). Then the protein concentration were measured with BCA assay, the final concentration is about 9mg/ml.
We tested the enzyme activity with the substrate of 0.5mM ABTS. ABTS is a light-green liquid reagent, laccase can oxidize the substrate and turn it into a dark green(C) to purple color(D). We measured the enzyme activity curves by testing OD562 and reaction time(D).
Updated by NPU - CHINA 2022
Laccases are multi-copper-containing proteins that catalyze the oxidation of phenolic substrates with concomitant reduction of molecular oxygen to water.We found that CotA seemed to degrade PAHs, so we made the following attempts.
Fig.1. SDS-PAGE analysis of AE5 mutant strain CotA . SDS-PAGE was used to analyze the expression of CotA. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ , respectively. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. . Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.
We changed the temperature and the concentration of the inducer. The figure above clearly shows that the target protein induced in LB medium is present in the supernatant (Fig. 1).
Fig.2. SDS-PAGE analysis of AE5 mutant strain CotA.SDS-PAGE was used to analyze the expression of CotA . Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ , respectively. The pellet was then dissolved in MSM medium without IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli . Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet.
After 2 d of induction in MSM medium without IPTG, the target proteins CotA can be clearly visualized in supernatant fraction of the whole bacterial lysate, which proves that the engineering iteration is effective (Fig. 2).
Fig.3. Oxidation of phenanthrene with the whole bacteria of CotA and LipH8 at 20 ℃ for 1 d, 3d, and 5d. The experiment was carried out in MSM medium without IPTG, and the oxidation was determined using noncellular components as the control. The differences in the PAH oxidation were determined by comparing the controls based on one-way ANOVA followed by Dunnett’s test (* P < 0.05).
SDS-PAGE results showed that the constructed expression system was successful. In order to verify whether the protein had biological activity, the concentration of phenanthrene was detected by HPLC. Both CotA and LipH8 could degrade phenanthrene, and coexpression of CotA and LipH8 was more effective (Fig. 3).
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