PARTNERSHIP

Starting from April, IvyMaker-China and ASTWS-China has had virtual meetups every 1-3 weeks to have discussion about progress, problems they met in the framework of RESEARCH, HUMAN PRACTICES, EDUCATION AND COMMUNICATION and MODEL. They have set up a long-term, in-depth, mutual partnership through the process.

April 3rd:
Confirm subject intention (PET degradation), potential of partnership and its framework (research, wet lab, human practice, education and communication, model)
April 10th:
Share preliminary research results; confirm the key enzyme (PETase and MHETase)
April 24th:
Share project design; Confirm host and etc.,
May 8th:
Discuss about the human practice framework and possibility of joint-interview
June 5th:
Share and discuss about progress of human practice activity and feedback
June 19th:
Share and discuss about plans of model construction
July 3rd:
Share and discuss about progress and feedback of human practice activity; also wet lab
July 24th:
Co-launch the Gene Hacker Lecture Series, organize and arrange the promotion
August 7th:
Share and discuss about progress and feedback of human practice activity; also wet lab
August 14th:
Share and discuss about the progress of wet lab; Check details about the Gene Hacker Lecture
August 22nd:
The day of Gene Hacker Lecture
September 4th:
Share and discuss about progress and feedback of human practice activity, model and wet lab
September 25th:
Share and discuss about progress and feedback of model and project implementation
October 2nd:
Organize all the data we have conducted

We had meeting every 1-3 weeks with ASTWS-China to share our progress in wet lab, discuss problems and provide mutual support. For example:
ASTWS-China planned to use plastic fragments as substrate which turns out not responding well, IvyMaker-China shipped PET powder we used to them to continue experiment.

Figure 1: PET powder sample IvyMaker-China mailed to ASTWS-China
Using the same test PET powder, IvyMaker-China and ASTWS-China did a comparison test of the enzyme activity efficiency between surface display system and free enzyme.

IvyMaker-China:

Determine the Michaelis constant of PETase and select p-nitrophenyl acetate as the substrate; At the same time, explore whether the addition of MHETase will affect the kinetic parameters of PETase. The determination method is as follows:

PETase kinetic parameter determination method:
Take 0.2, 0.5, 1, 2, 3, 4 mm p-nitrophenyl acetate as substrate, OD600 as 1, buffer 45 mm Na₂HPO₄ HCl (pH 7.0), 90 mM NaCl, and 10% (V /V) DMSO for 90s at 30°C, 900 rpm, 1 ml of reaction system, 15000 g centrifugation for 1 min, take the supernatant and stop the reaction, and determine the absorption value at 415nm by enzyme labeling instrument at room temperature.

Determination method of kinetic parameters of PETase mixed MHETase:
The method is the same as above, in which the OD600 of PETase and MHETase is 1.
According to the measured results, the Michaelis Menten equation was fitted with graphpad prism. The results are as follows. It is calculated that the Vmax of PETase is 14.67μm/s, while the Vmax of mixed MHETase is 16.5μm/s, which is 12.5 higher than that of PETase. The results show that adding MHETase can improve the activity of PETase to a certain extent when p-nitrophenyl acetate is used as substrate.

Figure 2: the Michaelis equation fitting curve of PETase (left); Figure 3: the Michaelis equation's fitting curve of PETase + MHETase

ASTWS-China:

They selected p-Nitrophenylbutyrate (p-NPB) as the substrate, which can be hydrolyzed by PETase to p-nitrophenol (p-NP). Using p-NP assay, the enzyme activity of PETase and the impact of the addition of MHETase can be characterized by measuring the optimal optical density (OD) values at 405 nm. While overexpressing the PETase protein and PETase+MHETase dual enzymes, respectively, the results indicated that the addition of MHETase could help to increase the OD value of p-NP to some extent.

Figure 4: OD405 of p-NPB (0, 0.5, 1, 2, 4, 8 mM) hydrolysisby overexpressed PETsae-SC+OmpR and PETase-SC + OmpR + MHETase-ST, respectively.

Conclusion:

Compared with the data obtained by two teams independently, it is found that the addition of MHETase could help to improve the enzymatic activity of PETase to some extent.

We had continuous communication with ASTWS-China. We will have a monthly virtual meetup to share the feedback of our HP activities for each other's reference to further improve the project. For example, ASTWS-China has shared us their interview experience with a local Sewage Plant, and we shared with them feedback gained from attending CCiC8.

We lauched an online public education and communication lecture called "GENE HACKER" with ASTWS-China, Worldshaper-Nanjing and Worldshaper-Shanghai. We invited highschoolers and their familes interested in synthetic biology or bio in genreal to join this event and shared background info and lab design of our projects.

We had several meetups with ASTWS-China to explore the potential of the model design, problems we met in process.

Team:IvyMaker-China/Partnership