Characterization - Standard Tracks: ULaverne_Collab decided to characterize three plastic degrading enzyme parts. The parts characterized were: PETase (BBa_K2651000), Mutant PETase (BBa_K2651001), and MHetase (BBa_K2651002). These three parts were ran on an SDS page in order to evaluate molecular weight in kDa. The three parts were ran along with the protein ladder as well as a negative control, which in this case was the fluorescence protein Amil CP. Each part has Amil CP within the construct so if there is an increase of protein expression in comparison to the control, the specific enzyme can be further characterized. The exepcted kDa for PETase and Mutant PETase was 28.6 kDa. The expected kDa of MHetase differed with a kDa of 62.8. The resulting gel can be seen below:
Figure 1. SDS page showing Amil CP (Negative control), and the kDa of PETase, Mutant PETase, and MHetase.
The SDS page did not have clear bands, but the kDa value estimates were still taken to be where the band should be. These molecular weights were used to compare to the negative control in order to obtain a relative density through the use of the ImageJ program. The expected molecular weight for each part was highlighted with a box and the same size box was put around the negative control at the same molecular weight. This was done for each part individually in order to obtain an accurate value of relative density in relation to each band. These relative densities indicate the fold change of expression and were graphed as seen below:
Figure 2. Bar Graph showing relative densities for Amil CP (used as the standard), PETase, Mutant PETase, and Mhetase.
The relative density of each part was close to 1, which was the standard value. This value explains that the PETase, Mutant PETase, and MHetase did not express any protein other than the Amil CP. The relative density for each part is as follows: Amil CP = 1, PETase = 1.04, Mutant PETase = 1.02, and MHetase = 1.14. These relative densities showed close to no fold change of expression and the construct was a false positive once it turned blue. These parts most likely did not have the necessary protein in order to purify as the expected molecular weight did not have an expression increase of more than 1. Thi part most likely needs to be updated with a tag for purification in order to characterize it further.
Simplified List of Characterized Parts
- PETase (BBa_K2651000)
- Mutant PETase (BBa_K2651001)
- MHetase (BBa_K2651002)
