Difference between revisions of "Part:BBa K2940007"
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<partinfo>BBa_K2940007 short</partinfo>
 
<partinfo>BBa_K2940007 short</partinfo>
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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.
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===Usage and Biology===
 
===Usage and Biology===
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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.
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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.
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K2940007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2940007 SequenceAndFeatures</partinfo>
  
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==Characterization==
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We successfully assembled pSB1C3: DyP plasmid and transformed it into BL21 E. coli cells.
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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.
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<center>
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[[Image:T--Edinburgh_OG--TinaSDSGel.jpg|250px|Figure 1]]
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Figure 1. Protein expression of DyP and DyP-Cytb5 examined via SDS-PAGE. Visible protein bands at 45 kDa are shown in the image. 
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</center>
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Secreted DyP enzyme activity assay - ABTS assay 
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The secreted DyP enzymatic activity was also tested with ABTS assay.  The results showed the ABTS reagent without addition of hydrogen peroxide, the absorbance value of the blank changed by X, while the ABTS reagent with addition of hydrogen peroxide changed drastically by X. Due to lack of time, we didn’t achieved any conclusion 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. 
  
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For next steps 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. 
  
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==Reference==
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[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.
  
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[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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<!-- Uncomment this to enable Functional Parameter display  

Revision as of 18:24, 21 October 2019


Secreted dye-degrading peroxidase（DyP) 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.

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]
    Unknown
  • 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

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

Secreted DyP enzyme activity assay - ABTS assay 

The secreted DyP enzymatic activity was also tested with ABTS assay. The results showed the ABTS reagent without addition of hydrogen peroxide, the absorbance value of the blank changed by X, while the ABTS reagent with addition of hydrogen peroxide changed drastically by X. Due to lack of time, we didn’t achieved any conclusion 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 steps 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.