Protein extraction of WT PETase and mutant PETase, S245I from E. coli Results: PETase was successfully extracted.
Determination of protein concentration using Bradford assay Results: Resulting protein concentration: S245I PETase: 2.49 µg/µL Wild Type PETase: 2.08 µg/µL
SDS-PAGE for S245I PETase and WT PETase Result: Both Wild Type and S245I PETase are successfully expressed and purified.
Bacterial transformation Transformation of MHETase, WT PETase-MHETase, and S245I PETase-MHETase sequence within PET21b vector into E. coli C41(DE3) and TOP10 competent cells by heat shock of 42°C Method Results: Colonies were observed on the LB-carbenicillin plates. Recombinant plasmids were successfully transformed into the competent cells Colony PCR were then performed to verify the presence of insert DNA. Results were obtained on 10/02.
PET film digestion at 30°C for 96 hours 4µg and 9µg of purified protein, WT PETase and S245I PETase were used to digest PET films. Results: see SEM and HPLC data below
Digested PET film processing Results: PET film is ready for viewing under SEM.
Examination of digested PET films under SEM Results: The surface of PET after the digestion using 9µg of PETase mutant S245I, showing significant pitting present on the surface:
The surface of PET after the digestion using 9µg of wild type PETase, showing the pitting present on the surface:
The surface of PET in the absence of PETase, showing no visible evidence of enzymatic digestion:
Conclusion: The pitting resulting from the digestion of S245I PETase is much more significant than from the digestion of WT PETase. S245I PETase exhibits a higher PET depolymerization activity.
HPLC of solution from digestion of PET film Results: HPLC profile of solution resulted from PET film digestion using wild type PETase as the only enzyme:
HPLC profile of solution resulted from PET film digestion using S245I PETase as the only enzyme:
The retention time of TPA and MHET HPLC standards were at 4.64 minutes and 5.17 minutes respectively. Concentration of MHET intermediates and TPA monomers in solutions:
Conclusion: Resulting concentration of monomers was higher when PET film is digested by S245I PETase than by WT PETase. S245I PETase exhibits a higher depolymerization activity. Also, considerable amount of intermediate product, MHET were detected in PET digestion eluents suggested PET hydrolysis by single enzyme, WT PETase and S245I PETase was incomplete.
Colony PCR used MHETase primer Results: We confirmed that the experiment done on 28/12 is successful. The recombinant plasmids were transformed into C41 and TOP10 competent cells Results: We successfully amplified a PCR product of 498 bp in bacterial colonies transformed with recombinant plasmids containing MHETase, WT PETase-MHETase and S245I PETase-MHETase. Colony PCR of constructs transformed into C41(DE3) competent cells:
Colony PCR of constructs transformed into TOP10 competent cells:
Interview with Professor Jacky Ngo, CUHK
Interview with Professor T.F. Chan, CUHK
Protein extraction of WT PETase, S245I PETase (1) and (2), MHETase, WT PETase-MHETase and S245I PETase-MHETase Results: Protein is successfully extracted.
Interview with Mr. Sidhant Gupta and Mr. Utkarsh Goel, Clearbot
SDS-PAGE of extracted protein constructs on 05/05/21 Results:
Bradford Assay of WT PETase, S245I PETase (1) and MHETase Results:
PET film digestion at 30°C for 96 hours In the enzyme cocktails, 4μg WT PETase or S245I PETase were mixed with 4μg and 8 μg MHETase. A solution with only 4μg WT PETase and a solution with only S245I PETase was used as a control to compare the PET degradation rate of single-enzyme systems and dual-enzymes systems.
Protein extraction of WT PETase, S245I PETase and WT PETase-MHETase chimera Results: Protein is successfully extracted and we got E1, E2. We are going to perform SDS-PAGE with these.
SDS PAGE of extracted proteins from 17/07/21 Results: WT PETase-MHETase chimera failed to express in the SDS-PAGE using the old extraction method. The old method needs to be optimized in order to extract the protein. Fractionation may be considered to extract it.
Digested PET film processing PET film digestion using enzyme cocktails on 16/07/21 Results: The eluent is ready for HPLC.
Large scale protein extraction (400mL bacterial culture) Our chimeric proteins, S245I PETase-MHETase and PETase-MHETase, were unable to be extracted using old method. The protocol was revised to scale up the volume of bacterial culture to 400mL, lower the temperature from room temperature to 16°C and lengthen the incubation time from 18h to 30h. A 4 mL starter culture of S245I PETase-MHETase and WT PETase-MHETase were added to a 400mL LC medium. The mixture was shaken at 37°C until OD600 = 0.8. 0.5mM IPTG was then added for induction. After adding IPTG, the mixture was shaken at 16°C for 30h. After induction, 400 mL bacterial culture was divided into two 200 mL solutions and harvested the cells by centrifugation at 5,000 rpm at 4°C for 15 minutes. The cell pellets were resuspended by adding 20 mL of lysis buffer. We further sonicated the suspension 5 times for 15 cycles; each cycle consists of 10s with sonication followed by 10s without sonication. The sonication power is 6-8 W. After centrifuging at 13,000 rpm for 20 min at 4°C, 20mL supernatants were collected. 2 mL Ni-NTA resin was washed with lysis buffer for 5 times with short spins at 3,000 rpm and kept on ice. Then, 10 mL of the supernatant was mixed with 1 mL Ni-NTA resin and then shook on ice for 1 hour at 50 rpm. After rinsing the Nickel column 3 – 4 times with lysis buffer, the mixture was loaded to a column. The column was then washed 3 times with wash buffer and 3 times with 2 mL elution buffer. For SDS-PAGE, we mixed 15 µL of purified proteins and 5 µL 4× loading dye and loaded into the wells.
HPLC of solutions after PET film digestion using enzyme cocktails Results:
HPLC profile of products released from PET film digestion using 4 μg WT PETase and 4 μg MHETase Maximum intensity: 1736.7 cps at 4.64 min
HPLC profile of products released from PET film digestion using 4 μg WT PETase and 8 μg MHETase Maximum intensity: 3986.7 cps at 4.62 min
HPLC profile of products released from PET film digestion using 4 μg WT PETase Maximum intensity: 16000 cps at 4.63 min
HPLC profile of products released from PET film digestion using 4 μg S245I PETase and 4 μg MHETase Maximum intensity: 7756.7 cps at 4.63 min
HPLC profile of products released from PET film digestion using 4 μg S245I PETase and 8 μg MHETase Maximum intensity: 8166.7 cps at 4.62 min
HPLC profile of products released from PET film digestion using 4 μg S245I PETase Maximum intensity: 4906.7 cps at 4.63 min
HPLC profile of products released from PET film digestion without using enzyme (negative control) Maximum intensity: 2193.3 cps at 4.63 min
Conclusion: The enzyme cocktail with S245I PETase and MHETase is successful. Furthermore, a mixture of PETase and MHETase in a 1:2 ratio shows better degradation activity than a mixture in a 1:1 ratio. With the same ratio, mixtures containing S245I PETase have a higher degradation activity than mixtures containing wild type PETase. Additionally, mixtures with MHETase have a higher degradation activity than mixtures that do not contain MHETase.
SDS PAGE of extracted proteins from 21 – 24/7/21 Results:
Conclusion: WT PETase-MHETase and S245I PETase-MHETase were successfully expressed and purified using new protein extraction protocol (400mL bacterial culture) but there were a few extra bands. Size of WT PETase and S245I PETase: 30kDa Size of MHETase: 65kDa Size of WT PETase-MHETase and S245I PETase-MHETase: 95kDa
PET film digestion We immersed our 6mm×6mm PET films into solutions containing 16μL chimeric proteins, WT PETase-MHETase and S245I PETase-MHETase, as well as MHETase only and the negative control. We incubated the solutions for 96h in 30°C.
PET film processing
HPLC of solutions after PET film digestion using chimeric and single enzymes
Interview with Ms. Dana Winograd, Plastic Free Seas
Sharing session by Ms. Dana Winograd, Plastic Free Seas
iGEM Symposium 2021, HKUST
SEM We examined the digested PET film using 16μL chimeric proteins under the SEM. Results: The surface of PET after the digestion of 16μL of MHETase, showing pitting of PET film surface:
The surface of PET after the digestion of 16μL of mutant S245I PETase-MHETase chimera, showing pitting of PET film surface:
The surface of PET after the digestion of 16μL of WT PETase-MHETase chimera, showing pitting of PET film surface:
Negative control: the surface of PET in the absence of enzyme, showing no visible evidence of enzymatic digestion:
SDS-PAGE and Western blot Since a few bands were obtained in purified chimeric proteins, WT PETase-MHETase and S245I PETase-MHETase, SDS-PAGE and western blot were performed to confirm the identity of purified proteins. Both chimeric proteins were cloned into the expression vector pET-21b (+) which has a C-terminal His-Tag, therefore, anti-His Tag antibody was used for verification.
Lane 1: WT PETase Lane 2: S245I PETase Lane 3: MHETase Lane 4: WT PETase-MHETase Lane 5: S245I PETase-MHETase
iGEM sharing session for G7 and G8 GT school students
Interview with Mr. Nigel Mattraver, New Life Plastics
Interview with BioArchitect, HKSTP
Hong Kong High School iGEM Symposium 2021
