Difference between revisions of "Part:BBa K2684000"

Latest revision as of 03:45, 2 October 2024

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.

Contributed by RDFZ-CHINA 2024

Construction of BsLac and INP-BsLac Engineered Strains

Objective and Methods

To construct the BsLac and INP-BsLac engineered strains, the gene encoding BsLac was first synthesized and optimized for E. coli codons. For the INP-BsLac fusion strain, a truncated ice nucleation protein (INP) sequence was synthesized and placed upstream of the BsLac gene to create the fusion protein sequence. Both constructs were cloned into the pET23b plasmid using NdeI and XhoI restriction sites, with the T7 promoter controlling the expression of the downstream genes. The recombinant plasmids were synthesized and verified through sequencing (Azenta, USA; Genewiz, China).

Following successful sequencing verification, the plasmids were extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmids were then transformed into E. coli DH5α for plasmid storage and into E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C to ensure proper growth and plasmid maintenance.

Figure 1. Gel Electrophoresis of BsLac and INP-BsLac Gene Constructs

Results and Conclusion

The gel electrophoresis results confirm the successful construction of both strains:BsLac: 1542 bp；INP-BsLac: 2079 bp.

These results validate the successful cloning and setup of both BsLac and INP-BsLac engineered strains, enabling further studies on protein expression and function.

Analysis of Laccase Activity in Crude Enzyme Extracts from Engineered Strains

Objective and Methods

The objective of this experiment was to measure the laccase (BsLac) activity in crude enzyme extracts from engineered E. coli strains to verify successful expression and catalytic function.

1. Culture and Extraction: The engineered strains were cultured overnight at 37°C. After centrifuging 2 mL of the culture at 8000 rpm for 10 minutes, the cell pellet was resuspended in PBS (pH 7.4) and disrupted by ultrasonication to obtain crude enzyme extracts.

2. Laccase Activity Assay: Laccase activity was measured using the ABTS oxidation method. The reaction mix included 930 μL Na₂HPO₄-citric acid buffer (50 mM, pH 3.2), 20 μL ABTS (10 mM), and 50 μL enzyme extract. Absorbance at 420 nm was recorded for 1 minute to calculate enzyme activity.

3. Reaction Principle: ABTS stock solution is deep blue, which becomes blue-purple upon dilution. When oxidized by laccase, the solution changes from blue to green or blue-green, with strong absorbance at 420 nm.

Figure 2. Laccase Activity Assay for BL21-pET23b-BsLac Strain

Results and Conclusion

The engineered strain (BL21-pET23b-BsLac) showed significantly higher absorbance at 420 nm compared to control strains (BL21 and BL21-pET23b), confirming active BsLac expression. The higher laccase activity in the engineered strain suggests efficient oxidation of ABTS, as evidenced by the color change to green/blue-green.This indicates that the BsLac strain has strong laccase activity, supporting its potential for environmental applications like oil degradation.

The BsLac-engineered strain demonstrated significantly enhanced laccase activity, confirming its ability to oxidize ABTS efficiently, and highlighting its application potential in environmental remediation.

pH and Temperature Effects on Laccase Activity

Objective and Methods

The aim of this experiment was to determine the optimal pH and temperature for laccase (BsLac) activity, providing insights for its application. Laccase activity was measured at 30°C across a pH range of 2.0 to 6.0, using glycine-HCl buffer for pH 2.0 and Na₂HPO₄-citric acid buffer for pH 3.0–6.0. To assess the temperature effect, activity was measured from 20°C to 60°C in Na₂HPO₄-citric acid buffer (pH 3.2), using ABTS as the substrate.

Figure 3. pH and Temperature Effects on Laccase Activity

Results and Conclusion

Laccase activity was highest at pH 3.2 and peaked at 40°C. Activity decreased significantly at lower and higher pH values, and temperatures above 40°C caused a marked decline, indicating enzyme instability. These findings highlight that BsLac is most active under moderately acidic conditions and at 40°C, guiding its potential use in biotechnological applications.

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.

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).