Difference between revisions of "Part:BBa K2940007"

Latest revision as of 02:10, 22 October 2019

Secreted dye-degrading peroxidase（DyP)

This part is designed as the control group to check whether the original DyP can be expressed and degrade dyes. So the coding sequence was referenced from NCBI (NC_000964.3) without any modification. It was cloned into pSB1C3 using classical cloning and was verified using SDS-PAGE analysis and ABTS assay.

Usage and Biology

Dye-decolorising peroxidase (DyP) has been recognised for its capacity to catalyse extracellularly the H2O2-dependent oxidation of various dye molecules, including anthraquinone-based dyes and azo dyes [1]. The DyP chosen here is from Bacillus subtilis (BsDyP) and recent study showed this peroxidase can maintain activity in an environment at high temperature (53h ± 11h at 40 °C) [2], suggesting its high stability in extreme working environment.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 10
Illegal NheI site found at 33
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 155
Illegal BsaI site found at 164
Illegal BsaI site found at 1130
Illegal SapI site found at 560
Illegal SapI site found at 918
Illegal SapI.rc site found at 1074

Characterization

We successfully assembled pSB1C3: DyP plasmid and transformed it into BL21 E. coli cells.

The SDS-PAGE analysis of the crude extract cells of peroxidases shows that the supplement of hemin in the culture medium facilitated the formation of a stronger band at around 45 kDa for both DyP and DyP-Cytb5 (Figure 1). However, similar bands were also shown in cell crude extracts without hemin induction and some bands were also disappearing upon induction. Unfortunately, the SDS-PAGE analysis was not repeated to confirm the protein expression due to the time limit.

Figure 1. Protein expression of DyP and DyP-Cytb5 examined via SDS-PAGE. Visible protein bands at 45 kDa are shown in the image. 

The secreted DyP enzymatic activity was tested with ABTS assay. When adding hydrogen peroxide into the ABTS reaction mixture to test the OD420nm, the absorption value of the blank (adopting water instead of the extract cells) varied from 0 to 1 within 30 minutes, likely due to the reaction between hydrogen peroxide and ABTS. Next, ABTS assay was repeated, but no hydrogen peroxide was added at this time , and the absorbance value of the blank changed very small, while the absorbance value of the whole cells and extract cells group with the additional H2O2 was better than that of H2O2-free group, left H2O2-free samples overnight and on the next day the value of extract cells rose while the absorbance value of whole-cells fell, but it was not triplicated. Hence, as the time is limited, any conclusion was not drawn on whether the secreted DyP revealed the enzyme activity via the ABTS test and whether H2O2 should be increased as a co-factor in the ABTS test.

For next step is required to optimize the ABTS protocol, to test secreted DyP and cellular envelopes with immobilized DyP enzymatic activity. It would be interest to explore until what extent the hydrogen peroxide reacts with ABTS, adjusting the variables that control the reaction. As well, to explore other reagents that present activity in presence of hydrogen peroxide for replacing the ABTS.

Reference

[1] E. Van Bloois, D. E. Torres Pazmiño, R. T. Winter, et al., “A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily,” Applied Microbiology and Biotechnology, vol. 86, no. 5, pp. 1419–1430, 2010.

[2] A. Santos, S. Mendes, V. Brissos, et al., “New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: Towards biotechnological applications,” Applied Microbiology Biotechnology, vol. 98, no. 5, pp. 2053–2065, 2014.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2940007 short</partinfo>
+This part is designed as the control group to check whether the original DyP can be expressed and degrade dyes. So the coding sequence was referenced from NCBI (NC_000964.3) without any modification. It was cloned into pSB1C3 using classical cloning and was verified using SDS-PAGE analysis and ABTS assay.
 ===Usage and Biology===
-Dye-degrading peroxidase (DyP) is from Bacillus subtilis. This part is designed as the control group to check whether the original DyP can be expressed and degrade dyes. So the coding sequence was referenced from NCBI (NC_000964.3) without any modification. (NC_000964.3)  It was cloned into pSB1C3 using classical cloning and was verified using SDS-PAGE analysis and ABTS assay.
+Dye-decolorising peroxidase (DyP) has been recognised for its capacity to catalyse extracellularly the H2O2-dependent oxidation of various dye molecules, including anthraquinone-based dyes and azo dyes [1]. The DyP chosen here is from Bacillus subtilis (BsDyP) and recent study showed this peroxidase can maintain activity in an environment at high temperature (53h ± 11h at 40 °C) [2], suggesting its high stability in extreme working environment.
 ===Sequence and Features===
 <partinfo>BBa_K2940007 SequenceAndFeatures</partinfo>
+==Characterization==
+We successfully assembled pSB1C3: DyP plasmid and transformed it into BL21 E. coli cells.
+The SDS-PAGE analysis of the crude extract cells of peroxidases shows that the supplement of hemin in the culture medium facilitated the formation of a stronger band at around 45 kDa for both DyP and DyP-Cytb5 (Figure 1).  However, similar bands were also shown in cell crude extracts without hemin induction and some bands were also disappearing upon induction. Unfortunately, the SDS-PAGE analysis was not repeated to confirm the protein expression due to the time limit.
+<center>
+[[Image:T--Edinburgh_OG--TinaSDSGel.jpg|250px|Figure 1]]
+Figure 1. Protein expression of DyP and DyP-Cytb5 examined via SDS-PAGE. Visible protein bands at 45 kDa are shown in the image.
+</center>
+The secreted DyP enzymatic activity was tested with ABTS assay. When adding hydrogen peroxide into the ABTS reaction mixture to test the OD420nm, the absorption value of the blank (adopting water instead of the extract cells) varied from 0 to 1 within 30 minutes, likely due to the reaction between hydrogen peroxide and ABTS. Next, ABTS assay was repeated, but no hydrogen peroxide was added at this time , and the absorbance value of the blank changed very small, while the absorbance value of the whole cells and extract cells group with the additional H2O2 was better than that of H2O2-free group, left H2O2-free samples overnight and on the next day the value of extract cells rose while the absorbance value of whole-cells fell, but it was not triplicated. Hence, as the time is limited, any conclusion was not drawn on whether the secreted DyP revealed the enzyme activity via the ABTS test and whether H2O2 should be increased as a co-factor in the ABTS test.
+For next step is required to optimize the ABTS protocol, to test secreted DyP and cellular envelopes with immobilized DyP enzymatic activity. It would be interest to explore until what extent the hydrogen peroxide reacts with ABTS, adjusting the variables that control the reaction. As well, to explore other reagents that present activity in presence of hydrogen peroxide for replacing the ABTS.
+==Reference==
+[1] E. Van Bloois, D. E. Torres Pazmiño, R. T. Winter, et al., “A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily,” Applied Microbiology and Biotechnology, vol. 86, no. 5, pp. 1419–1430, 2010.
+[2] A. Santos, S. Mendes, V. Brissos, et al., “New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: Towards biotechnological applications,” Applied Microbiology Biotechnology, vol. 98, no. 5, pp. 2053–2065, 2014.
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