Difference between revisions of "Part:BBa K3576001"

(Contribution:Bioplus-China 2022)
(Contribution:Bioplus-China 2022)
Line 34: Line 34:
 
==Contribution:Bioplus-China 2022==
 
==Contribution:Bioplus-China 2022==
  
[[File: conresult2.png|500px|thumb|center|Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.]]
+
[[https://static.igem.wiki/teams/4413/wiki/experiment/conresult2.png|500px|thumb|center|Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.]]

Revision as of 08:10, 28 July 2022


PETase expression system

It is the key part that is responsible for expressing PETase. The PETase can hydrolyze PET to MHET. In order to ensure that PETase can be fully in contact with extracellular PET. The sequence of coding pelB signal peptide was added before PETase.

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 (40 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.




Contribution:Bioplus-China 2022

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