Current Stage of Results:
- We were able to successfully add our parts to our e.coli
- Still in the process of testing and looking for actual degradation.
- Aim to take SEM images to look for nano sized missing pockets on the plastics surface.
- Figure 1: PCR products creating overlapping ends for the Gibson assembly of our parts. K1357009 is our amilCP and double terminator part at 874 bp. The two PETase enzymes are around 1000 bp each with slight variation because of the difference in overlapping ends between parts. mHETase is around 2000 bp and the promoter and vector is also near 2000.
- Figure 2: Plates showing blue colonies on plates for each of our circuits, proving that amilCP reporter is active and that the DNA has been assembled properly.
- Figure 3: Assembled parts were digested to ensure proper DNA lengths in the circuits. The first gel shows the mHETase circuit digested once in lane one which made a band at 6000 bp, and once digested with two enzymes showed bands at 2000 bp for the vector and 4000 bp for the assembled circuit of mHETase, mutPETase, and the k1357009 reporter. The second and third gels show the PETase circuits digested with one enzyme at about 4000 bp, and then when digested with two it showed two bands very near to 2000 bp which is where the vector and the assembled circuit of the PETase and k1357009 will be.
- Figure 4: After assembly and transformation, PCR reactions were done on the mini prepped plasmids to ensure that the circuits contained the proper enzymes. In order from left to right, the mutPETase from mHET circuit, control PETase from control circuit, mHETase from mHETase circuit, and mutPETase from mutPETase circuit.
These results conclude that we cannot utilize this method to degrade plastic at real time with the current rate of the mutant PETase. We aim to further characterize with SEM to understand if the Mutant PETase is better than the Wild Type.