Difference between revisions of "Part:BBa K4724010"

 
 
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<partinfo>BBa_K4724010 short</partinfo>
 
<partinfo>BBa_K4724010 short</partinfo>
  
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We turn glutamine into leucine. We chose for this mutation because it changed from hydrophilic amino acids to hydrophobic amino acids, promoting the formation of an active pocket hydrophobic environment.
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<i>Is</i>PETase is a hydrolase produced by Ideonella sakaiensis that can degrade PET. Q126L changes from hydrophilic to hydrophobic amino acids, leading to the formation of a hydrophobic environment in the active pocket.
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<h1>Constraction</h1>
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The plasmid was PCR with pET-22b(+) as the vector, and the PCR bands were verified by nucleic acid gel electrophoresis after mutation. The plasmid with the correct bands was transformed into <i>E.coli</i> BL21(DE3) sensory state.
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https://static.igem.wiki/teams/4724/wiki/fig-1.png
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Fig.1 Nucleic acid gel electrophoresis for PCR of plasmid
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<h1>Characterization</h1>
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After protein purification, enzymatic reactions were performed to measure enzyme activity. The substrate used was PET powder, which was broken down into TPA and MHET in the presence of an <i>Is</i>PETase mutant, and the volume of purified enzyme solution required for a 500 μL reaction system was determined based on protein concentration. The reaction was carried out at 30°C, 37°C and 45°C for 48 h. After the reaction, the reaction solution was analyzed by high performance liquid chromatography (HPLC), and the liquid phase result at 6 min corresponded to TPA and the liquid phase result at 8 min corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the product concentrations by standard curves, as shown in Fig.2
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<img src="https://static.igem.wiki/teams/4724/wiki/ab.png " style="width:60vw;">
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<img src="https://static.igem.wiki/teams/4724/wiki/cd.png " style="width:60vw;">
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<img src="https://static.igem.wiki/teams/4724/wiki/ef.png " style="width:60vw;">
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Fig.2 Concentrations of the products TPA and MHET of 500 nM WT and Q126L reacted with PET powder at different temperatures for 48h. (A) and (B) are the concentrations of the products obtained at a reaction temperature of 30°C; (C) and (D) are the concentrations of the products obtained at a reaction temperature of 37°C; (E) and (F) are the concentrations of the products obtained at a reaction temperature of 45°C. The concentrations of TPA and MHET were obtained at different temperatures.
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<h1>Conclusion</h1>
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At 30 °C, <i>Is</i>PETase<sup>Q126L</sup> activity decreased compared to WT.
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At 37 °C, <i>Is</i>PETase<sup>Q126L</sup> activity decreased compared to WT.
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At 45 °C, <i>Is</i>PETase<sup>Q126L</sup> product concentration is about 2 times that of WT.
  
  

Latest revision as of 14:51, 12 October 2023


IsPETaseQ126L-6xHis Tag

IsPETase is a hydrolase produced by Ideonella sakaiensis that can degrade PET. Q126L changes from hydrophilic to hydrophobic amino acids, leading to the formation of a hydrophobic environment in the active pocket.

Constraction

The plasmid was PCR with pET-22b(+) as the vector, and the PCR bands were verified by nucleic acid gel electrophoresis after mutation. The plasmid with the correct bands was transformed into E.coli BL21(DE3) sensory state. fig-1.png

Fig.1 Nucleic acid gel electrophoresis for PCR of plasmid

Characterization

After protein purification, enzymatic reactions were performed to measure enzyme activity. The substrate used was PET powder, which was broken down into TPA and MHET in the presence of an IsPETase mutant, and the volume of purified enzyme solution required for a 500 μL reaction system was determined based on protein concentration. The reaction was carried out at 30°C, 37°C and 45°C for 48 h. After the reaction, the reaction solution was analyzed by high performance liquid chromatography (HPLC), and the liquid phase result at 6 min corresponded to TPA and the liquid phase result at 8 min corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the product concentrations by standard curves, as shown in Fig.2

Fig.2 Concentrations of the products TPA and MHET of 500 nM WT and Q126L reacted with PET powder at different temperatures for 48h. (A) and (B) are the concentrations of the products obtained at a reaction temperature of 30°C; (C) and (D) are the concentrations of the products obtained at a reaction temperature of 37°C; (E) and (F) are the concentrations of the products obtained at a reaction temperature of 45°C. The concentrations of TPA and MHET were obtained at different temperatures.

Conclusion

At 30 °C, IsPETaseQ126L activity decreased compared to WT.

At 37 °C, IsPETaseQ126L activity decreased compared to WT.

At 45 °C, IsPETaseQ126L product concentration is about 2 times that of WT.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 56
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 56
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 790
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 56
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 56
    Illegal AgeI site found at 546
  • 1000
    COMPATIBLE WITH RFC[1000]