Our team aims to enhance the activity of the PETase enzyme by creating three specific amino acid mutations on PETase (IsPETase^{S121E/D186H/R280A}) under inducible promoters, T7 and pGal1,10, allowing us to express as many variants as desired. As a result, we successfully developed the following constructs: T7-IsPETase (BBa_K5094006), T7-IsPETase^Thr116Ala (BBa_K5094007), T7-IsPETase^{Thr116Ala/M154Thr} (BBa_K5094008), T7-IsPETase^{Thr116Ala/K259Glu} (BBa_K5094009), and pGal1,10-IsPETase (BBa_K5094010). We believe our project can contribute significantly to the community. For more detailed information, please refer to our experiments and parts pages.

Engineered parts

Using site-directed mutagenesis, our team created T7-IsPETase (BBa_K5094006), T7-IsPETase^Thr116Ala (BBa_K5094007), T7-IsPETase^{Thr116Ala/M154Thr} (BBa_K5094008), T7-IsPETase^{Thr116Ala/k259Glu} (BBa_K5094009). Our team also cloned IsPETase downstream of pGal1,10 promoter, creating pGal1,10-IsPETase (BBa_K5094010). The engineered parts can potentially be used for degrading PET plastic, and their degradability compared to PETase enzyme with other amino acid mutations can be further investigated for future teams.

T7 plasmid containing 6-his tag

The T7 promoter we used contains an N-terminal 34aa precursor peptide with a histidine tag sequence, 6-his tag, to bind to the nickel-resin column for the in vitro protein purification to extract the proteins our team is interested in (T7 Promoter System, 2016). Future teams could do further enzyme purification if using our plasmids. Also, with the 6-his tag, it was easy for us to find the antibodies for western blot functional assay.

Troubleshooting

Throughout the project, our team encountered several problems and used troubleshooting to resolve each issue. Below is a list of summaries for each problem, solutions to the problems, and possible applications to future iGEM projects. If other teams happen to encounter similar problems, feel free to use these as references. For more information, refer to parts and experiments page.

No Mutations for pGal1, 10 Promoter

For the IsPETase gene cloned downstream of the pGal1,10 promoter, our team couldn’t make any mutations on the IsPETase gene After talking to Dr. Hsiao-Fen Han from NTU, we realized that the problem is that the plasmid is too big. The best way to fix the situation is to add the mutations to the IsPETase gene sequence and ask external companies to synthesize them. Our team decided to use pGal1,10 promoter-eGFP plasmid as a control v.s pGal1,10 promoter-IsPETase plasmid for functional assays.

Incorrect Amino Acids Mutated

Our team did not mutate the correct regions on the T7-IsPETase gene, and instead created T7-IsPETase, T7-IsPETaseThr116A, T7-IsPETaseThr116A/k259Glu, T7-IsPETaseThr116A/M154Thr. We talked to Dr. Hsiao-Fen Han from NTU and realized that the IsPETase gene sequence contained multiple DNA fragments with repeat regions that our team didn’t expect while designing those mutations, leading to challenges for the site-directed mutagenesis technique. The primers containing mutant nucleotides that our team desired to switch specific amino acids targeted incorrect amino acids due to these repeats so our team created T7-IsPETase, T7-IsPETaseThr116A, T7-IsPETaseThr116A/k259Glu, T7-IsPETaseThr116A/M154Thr instead. One way to solve this problem is to add the mutations to the IsPETase gene sequence directly and order the synthesized mutant gene from the mission biotech company. After that, the team will do the traditional cloning as the team did with the wild-type IsPETase gene.

Education Materials

To promote synthetic biology and increase awareness of the problem of plastic pollution, our team organized educational lectures and laboratory workshops in our school. We hope to take action in environmental conservation by educating the public more about the severity of plastic pollution and related Sustainable Development Goals (SDGs). At the same time, we hope to spark interest in younger students to take part in research related to synthetic biology. The following lesson plans and lecture slides can be used as references for future iGEM teams to share knowledge regarding molecular biology.

SP side 7th-grade education workshop

IP and SP are the two different departments our school’s students are divided into. IP students focus more on applying to schools overseas while SP students use Taiwan’s curriculum. Because Taiwan’s curriculum still focuses quite heavily on tests, and things like SDGs or recycling plastic don’t appear that important in tests, the teachers basically “skip” the information due to the lack of importance. Also, with IP students being the majority of the people, our iGEM team contains very few SP students. We decided to combine the curriculum with our project to make a presentation that emphasizes the global plastic waste problem, how synthetic biology can help address the problem, and UNSDGs in Mandarin (the language SP learned their classes in)(our first language).

After discussing with the SP staff and science teachers, we decided to present to the 7th graders. We chose to include biomagnification, bioaccumulation, and food web, which are important concepts from their curriculum they already learned, and the idea of monomers and polymers, cloning, transcription, and translation which are important concepts from their curriculum they will be learning later on in our presentation. Using many interesting analogies that both the students and teachers love, it was quick and easy for the students to understand the severity of the microplastic problem and how our lab is going to fix it using synthetic biology.

This educational event caused a few students to go find the lecturer (Daniel Chang) and their teachers to ask more questions about the plastic problem and synthetic biology. This means some students became interested in iGEM due to this event, which is one of the goals we were aiming at in the first place.

SP Side 7^th-Grade Education Workshop Slide

Junior high students iGEM Synergy Workshop

This workshop is specifically designed for junior high students interested in iGEM but don’t have much background in doing molecular biology research. The students first got a brief introduction to molecular biology and cloning, then got an idea of what we really do in the iGEM lab. The students then learned about how to do bacterial inoculation and the reason to do so. At last, the students learned about how to use pipetmans and the way to dilute plasmids in the lab which was taught by competing in a “pipetman contest”.

After the event, we asked the students for their feedback on the event. On a scale of 1 to 5, for 1 being the lowest and 5 being the highest, we asked them how much they had learned about molecular biology, bacteria inoculation, and calculating concentration. 83.3 percent of the students believed that they have learned a lot during the workshop, rating the amount they learned as 4 or above. All of the students replied that they would be interested in learning more about molecular biology in the future and are interested in attending the iGEM competition. Our event successfully made more students experience the field of synthetic biology and provided an example for future teams to consider for educating more students.

Junior high students iGEM Synergy Workshop Slide

Citations

T7 Promoter System. (2016). Sigmaaldrich.com. https://doi.org/Dogri