Difference between revisions of "Part:BBa K4724006"
 
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We inserted glutamate E between amino acids 93 and 94 of <i>Is</i>PETase.We chose this site because the active pocket amino acids are changed, altering the tertiary structure of the protein.
 
We inserted glutamate E between amino acids 93 and 94 of <i>Is</i>PETase.We chose this site because the active pocket amino acids are changed, altering the tertiary structure of the protein.
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<h>Construction</h>
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<h1>Construction</h1>
 
The plasmid was subjected to PCR after mutation using pET-22b(+) as a vector, and Fig.1 shows the PCR bands of the plasmid verified by nucleic acid gel electrophoresis. The plasmid with correct bands was transformed into <i>E. coli</i> BL21(DE3) sensory state.  
 
The plasmid was subjected to PCR after mutation using pET-22b(+) as a vector, and Fig.1 shows the PCR bands of the plasmid verified by nucleic acid gel electrophoresis. The plasmid with correct bands was transformed into <i>E. coli</i> BL21(DE3) sensory state.  
 
https://static.igem.wiki/teams/4724/wiki/fig-1.png
 
https://static.igem.wiki/teams/4724/wiki/fig-1.png
  
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Fig.1 Plasmid PCR Nucleic Acid Gel Electrophoresis
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Fig.1 Nucleic acid gel electrophoresis for PCR of plasmid
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<h>Characterization</h>
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<h1>Characterization</h1>
 
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 6-min liquid phase result corresponded to TPA, and the 8-min liquid phase result corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the concentrations of the products by standard curves, as shown in Fig.2.
 
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 6-min liquid phase result corresponded to TPA, and the 8-min liquid phase result corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the concentrations of the products by standard curves, as shown in Fig.2.
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Fig.2 Concentrations of TPA and MHET, the products of 500nM WT and S93_I94insE reacted with PET powder for 48h at different temperatures. (A) and (B) are the concentrations of the products obtained at 30°C, (C) and (D) are the concentrations of the products obtained at 37°C, and (E) and (F) are the concentrations of the products obtained at 45°C.  
 
Fig.2 Concentrations of TPA and MHET, the products of 500nM WT and S93_I94insE reacted with PET powder for 48h at different temperatures. (A) and (B) are the concentrations of the products obtained at 30°C, (C) and (D) are the concentrations of the products obtained at 37°C, and (E) and (F) are the concentrations of the products obtained at 45°C.  
  
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<h>Conclusion</h>
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<h1>Conclusion</h1>
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At 30°C, the product of IsPETaseS93_I94insE was approximately 2-fold higher than that of WT; at 37°C, the product of IsPETaseS93_I94insE was approximately 2.5-fold higher than that of WT; and at 45°C, the product of IsPETaseS93_I94insE was approximately 9-fold higher than that of WT.
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At 30°C, the product of <i>Is</i>PETase<sup>S93_I94insE</sup> was approximately 2-fold higher than that of WT.
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At 37°C, the product of <i>Is</i>PETase<sup>S93_I94insE</sup> was approximately 2.5-fold higher than that of WT.
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At 45°C, the product of <i>Is</i>PETase<sup>S93_I94insE</sup> was approximately 9-fold higher than that of WT.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:45, 11 October 2023


IsPETaseS93_I94insE-6xHis Tag

We inserted glutamate E between amino acids 93 and 94 of IsPETase.We chose this site because the active pocket amino acids are changed, altering the tertiary structure of the protein.

Construction

The plasmid was subjected to PCR after mutation using pET-22b(+) as a vector, and Fig.1 shows the PCR bands of the plasmid verified by nucleic acid gel electrophoresis. The plasmid with 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 6-min liquid phase result corresponded to TPA, and the 8-min liquid phase result corresponded to MHET. The peak areas of the products outputted from the liquid chromatography were converted into the concentrations of the products by standard curves, as shown in Fig.2.

Fig.2 Concentrations of TPA and MHET, the products of 500nM WT and S93_I94insE reacted with PET powder for 48h at different temperatures. (A) and (B) are the concentrations of the products obtained at 30°C, (C) and (D) are the concentrations of the products obtained at 37°C, and (E) and (F) are the concentrations of the products obtained at 45°C.

Conclusion

At 30°C, the product of IsPETaseS93_I94insE was approximately 2-fold higher than that of WT.

At 37°C, the product of IsPETaseS93_I94insE was approximately 2.5-fold higher than that of WT.

At 45°C, the product of IsPETaseS93_I94insE was approximately 9-fold higher than 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 793
  • 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 549
  • 1000
    COMPATIBLE WITH RFC[1000]