Difference between revisions of "Part:BBa K3576001"
Line 20: Line 20:
 
The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm.  
 
The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm.  
 
As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.
 
As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.
−
[[File: PETase-Fig2.png|200px|thumb|center|Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.]]
+
[[File: PETase-Fig2.png|400px|thumb|center|Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.]]
  
  

Revision as of 06:34, 20 October 2020


PETase expression system

1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 97
    Illegal NgoMIV site found at 123
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 267

Results

1. Protein expression test

SDS-PAGE electrophoresis was used to check the expression of PETase protein. As shown in Figure 1, compared to the blank control, the lane contained PETase (42.3 KDa) protein indicated that this protein has been successfully expressed.

Figure 1 Protein SDS-PAGE electrophoresis results of PETase.

2. Enzyme Activity Test of PETase

The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm. As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.

Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.