Difference between revisions of "Part:BBa K5136027"

Latest revision as of 01:36, 2 October 2024

laccase-His tag

Biology

Laccase is a kind of Lignin-degrading enzyme. It is capable of oxidizing a wide range of substrates accompanied by the reduction of molecular oxygen to water (1). Moreover, extensive researches confirm that laccase can degrade and detoxify various pollutants with high efficiency at the laboratory scale. The enzyme we selected is isolated from Rheinheimera sp. (2).

Usage and Design

It can significantly improve parameters such as brightness, breaking length, tear coefficient, and bursting coefficient of recycled paper (3). In order to verify if it is capable of deinking waste paper, a His-tag (6×His) was added to the C-terminal of laccase for purification. We constructed this part and assembled it on the expression vector pET-28a(+).

Characterization

Agarose gel electrophoresis (AGE)
The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. Target bands (1659 bp) can be observed at the position between 1500 bp and 2000 bp(Figure 1).

Figure 1 DNA gel electrophoresis of the colony PCR products of BBa_K5136027_pET-28a(+) in E. coli BL21(DE3).

SDS-PAGE
The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM CuSO₄ and 0.03 mM IPTG at 28 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Figure 2), the target protein (62.6 kDa) can be observed at approximately 66 kDa on the purified protein lanes (FR).

Figure 2 SDS-PAGE analysis of laccase-His protein.

Deinking Experiments

A certain amount of purified enzyme solution was added to the pulp, and the pulp was filtered after 1 h reaction, and the gray value of the dried paper was measured to characterize the deinking effect of the enzyme. The higher the gray value, the better the deinking effect. As shown in Figure 3, laccase alone can also show a better deinking effect.

Figure 3 The gray value of paper cake deinked with laccase.

Reference

[1] J. Liu et al., Deciphering the alkaline stable mechanism of bacterial laccase from Bacillus pumilus by molecular dynamics simulation can improve the decolorization of textile dyes. Journal of Hazardous Materials 443, 130370 (2023).
[2] V. Gupta, S. Balda, N. Gupta, N. Capalash, P. Sharma, Functional substitution of domain 3 (T1 copper center) of a novel laccase with Cu ions. Int J Biol Macromol 123, 1052-1061 (2019).
[3] L. Arregui et al., Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact 18, 200 (2019).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 746
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 86
Illegal BamHI site found at 784
Illegal XhoI site found at 1655
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 746
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 746
Illegal AgeI site found at 175
Illegal AgeI site found at 506
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 ===Biology===
-Laccase is a kind of Lignin-degrading enzymes. It is capable of oxidizing a wide range of substrates accompanied by the reduction of molecular oxygen to water (1). Moreover, extensive researches confirm that laccase can degrade and detoxify various pollutants with high efficiency at the laboratory scale. The enzymes we choose is found from <b><i>Rheinheimera</I></b> <b>sp.</b>(2).
+Laccase is a kind of Lignin-degrading enzyme. It is capable of oxidizing a wide range of substrates accompanied by the reduction of molecular oxygen to water (1). Moreover, extensive researches confirm that laccase can degrade and detoxify various pollutants with high efficiency at the laboratory scale. The enzyme we selected is isolated from <i>Rheinheimera</i> sp. (2).
 ===Usage and Design===
-It can significantly improve parameters such as brightness, breaking length, tear coefficient and bursting coefficient of the recycled paper (3). In order to verify if it is capable to deink waste paper, a His-tag (6×his) was added to the C-terminal of laccase for purification. We constructed this part and assembled it on the expression vector pET-28a(+).
+It can significantly improve parameters such as brightness, breaking length, tear coefficient, and bursting coefficient of recycled paper (3). In order to verify if it is capable of deinking waste paper, a His-tag (6×His) was added to the C-terminal of laccase for purification. We constructed this part and assembled it on the expression vector pET-28a(+).
 ===Characterization===
 <b>Agarose gel electrophoresis (AGE)</b>
 <br>
-The constructed plasmids were transformed into <b><i>E. coli</I></b> BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. Target bands (1659 bp) can be observed at the position between 1500 bp and 2000 bp. (Figure 1)
+The constructed plasmids were transformed into <i>E. coli</i> BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. Target bands (1659 bp) can be observed at the position between 1500 bp and 2000 bp(Figure 1).
 <center><html><img src="https://static.igem.wiki/teams/5136/part/jrh/026colony.png" width="300px"></html> </center>
 <center><b>Figure 1  DNA gel electrophoresis of the colony PCR products of BBa_K5136027_pET-28a(+) in E. coli BL21(DE3). </b></center>
@@ Line 17: / Line 17: @@
 <b>SDS-PAGE</b>
 <br>
-The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM CuSO<sub>4</sub> and 0.03 mM IPTG at 28 °C GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Figure 2), the target protein (62.6 kDa) can be observed at the position around 66 kDa on the purified protein lanes (FR), although displayed with many other protein bands together.
+The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> BL21(DE3). After being cultivated and induced by 0.5 mM CuSO<sub>4</sub> and 0.03 mM IPTG at 28 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Figure 2), the target protein (62.6 kDa) can be observed at approximately 66 kDa on the purified protein lanes (FR).
 <center><html><img src="https://static.igem.wiki/teams/5136/part/jrh/027sds-page.png" width="300px"></html> </center>
-<center><b>Figure 2  SDS-PAGE analysis of laccase-his protein. </b></center>
+<center><b>Figure 2  SDS-PAGE analysis of laccase-His protein. </b></center>
 ===Deinking Experiments===
-A certain amount of purified enzyme solution was added to the pulp, and the pulp was filtered after 1h reaction, and the gray value of the dried paper was measured to characterize the deinking effect of the enzyme. The higher the gray value, the better the deinking effect. As shown in the figure 3, laccase alone can also show a better deinking effect.
+A certain amount of purified enzyme solution was added to the pulp, and the pulp was filtered after 1 h reaction, and the gray value of the dried paper was measured to characterize the deinking effect of the enzyme. The higher the gray value, the better the deinking effect. As shown in Figure 3, laccase alone can also show a better deinking effect.
 <center><html><img src="https://static.igem.wiki/teams/5136/part/jrh/process-0802-lac-na2sio3-vs-lac-na2sio3-0802.jpg" width="300px"></html> </center>
 <center><b>Figure 3  The gray value of paper cake deinked with laccase. </b></center>
 ===Reference===
+[1] J. Liu et al., Deciphering the alkaline stable mechanism of bacterial laccase from Bacillus pumilus by molecular dynamics simulation can improve the decolorization of textile dyes. <i>Journal of Hazardous Materials</i> <b>443</b>, 130370 (2023).
 <br>
-[1] J. Liu et al., Deciphering the alkaline stable mechanism of bacterial laccase from Bacillus pumilus by molecular dynamics simulation can improve the decolorization of textile dyes. Journal of Hazardous Materials 443, 130370 (2023).
+[2] V. Gupta, S. Balda, N. Gupta, N. Capalash, P. Sharma, Functional substitution of domain 3 (T1 copper center) of a novel laccase with Cu ions. <i>Int J Biol Macromol</i> <b>123</b>, 1052-1061 (2019).
 <br>
-[2] V. Gupta, S. Balda, N. Gupta, N. Capalash, P. Sharma, Functional substitution of domain 3 (T1 copper center) of a novel laccase with Cu ions. Int J Biol Macromol 123, 1052-1061 (2019).
+[3] L. Arregui et al., Laccases: structure, function, and potential application in water bioremediation. <i>Microb Cell Fact</i> <b>18</b>, 200 (2019).
-<br>
-[3] L. Arregui et al., Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact 18, 200 (2019).