Today marked our first day in the lab, and it felt completely different from any other lab experience we've had before.

Our lecturer, Ms. Ching, introduced us to the fundamentals of molecular biology, including DNA replication and retro-transcription. Having some prior knowledge of these topics helped us feel prepared for what lies ahead this year.

The rest of the session was to perform a bacterial transformation experiment on yeast competent cells. This was our first-ever molecular biology experiment, making it a truly new and exciting experience for us.

Our other lecturer: Ms. Parker Gave us an introduction to the field of synthetic biology.

Day 2 experiment: dividing plasmid from whole cell. We are doing this to extract the plasmids of our bacteria colonies we made last week.

• We use a base solution (NaOH solution) to remove the cell membrane and use phenol/chloroform to remove irrelevant proteins and rna.
• Proteins are broken by NaOA

Today the senior team from last year demonstrated the ways of Gel electrophoresis and PCR, two of the very important techniques we will be frequently using afterwards.

When they're done we kept on with the plasmid extraction protocol we were doing last week.

Today is the day when Ms. Parker announced the importance of notebooks, we will be taking notes on every IGEM activities. This notebook is essential for the judging in IGEM competition.

We were making a slides presentation of previous iGEM teams' project. This presentation gives us a better idea of IGEM competition and a big idea of our future path towards the IGEM Jamboree.

Later on, we when back to continue the experiment from last week, we measured the concentration of our plasmids in a spectrometer.

Today, we have a lecture on how PCR works and some PCR terms.

some terms we have learned:

1. Denaturation: the reaction that splits a dna double helix into two single strands.
2. Annealing: after the double helix is splitted, we add chosen primers with the single strands, and then, we lower the temperature for the primer to attach. (lowered temperature=50~65 c)
3. Extension: let the dna polymerase attach to complete the strands. (Dna polymerase is thermal bacteria’s Dna polymerase.)

After the lecture, we did some experiments on yeast genomic DNA extraction

Today we executed another important synthetic biology technique, PCR. we did PCR on the yeast genomic DNA we have extracted last week. After PCR, we were starting have a sense of the principle of synthetic biology.

PCR is the technique of amplifying a strain of gene x34000000000 times so that it will be easier to perform other analysis later.

Today we learned how to use NCBI and snapgene. Other than that, we finished the PCR protocol by running our data on NCBI.

Today, we learned how to do double enzyme digestion and understand the science behind restriction enzyme cut sites.

PCR

1. Use the GDNA (genomic DNA) previous made that are 100 ng/μl
2. Create master mix of 8 serves, (1 μl XMAI forward primer, 1 μl KPMII reverse primer , 8.5 μl dH2O, 12.5 μl 2x PCR)/ per serve
3. Further steps in protocol

Today we learned primer design using the software: Primer-C.

Such a website provides great convenience for people from students to biology experts, especially for students competing for competitions like iGEM.

Today we cloned of gene into pGal promoter, the pGal promoter is for yeast, we will be using T7 promoter for E.coli.

We also learned about the exclusiveness of promoters between species.

We want to multiply the amount of plasmids for each colony in order to see our ligation results, and keep the success samples to let it grow.

1. Separate a new agar plate with a marker into two sides
2. Use the pipet tip to touch a colony on the plate we did before, then spread them on the other agar plate. (to spread to bacteria)
3. Release the tip in an eppendorf tube containing the LB-AMP
4. Label number associated with the agar plate number
5. Repeat step 3~4
6. Further steps in protocol

Prior to the long vacation, our whole team has divided into three groups which came up with three independent project ideas. At the end the PET degradation team won the vote, the content of the other groups are also very interesting, one of them is about human health, achieved through a complicated procedure; while the other one is about modifying a type of tea plant and extracting's content to suppress dust mites.

The big thing of this month is bring up the progression of each group over the winter-break. We presented our progress and findings by groups (Wiki, Human-Practices and wetlab )

Our team project starts today!

We need pRSET A plasmid to clone the PETase gene for in vitro protein purification (purify protein into the tube). Despite being recently introduced to our project, Thanks to all the effort into training us, most of us can now properly follow protocols and understand molecular biology terms.

For the experiment part, we purified PETase protein with pRSET-A plasmid for in vitro purification. This process is more complicated than a lot of other experiments, it's because it involves tools and methods that are mew.(metal beads and resin)

Last week was just the preparation for protein purification, although there are only two steps remaining, it was finished successfully. After that we briefly discussed about our team aims and visions, Ms. Ching introduced the idea of site-directed-mutagenesis, the idea is to alter three amino acids of our PETase gene by altering three nucleotides of the gene, doing so will change the physical structure of our PETase and therefore theoretically enhance its ability of degrading PET.

We talked a little more about Site-directed mutagenesis, since all of us are still waiting for the materials for the next experiment. Hope the shipping can arrive next week.

While the human practice group rechecked contact with experts and other iGEM teams, they also confirmed schedules.

The wiki team started their search for good looking templates for our web page.

Today we once again did double enzyme digestion for pRSET-A plasmid, this has to be the most familiarized protocols we have ever done. Nothing went wrong except for me and Joseph losing our tubes.

For the HP team, OUR FIRST INSTAGRAM POSTS ARE OUT !!

Follow our Instagram @igem.kcisxiugangtaipei

Today we checked the wave lengths of our PET products, that pretty much all we did today.

Anyhow, we have the rest of the day to ourselves, to do our individually assigned work. We did paper works in the "experiments" document, and that's where the outline of our design cycle goes.

Today all of us made three sections of the plate covered with transformed or not transformed colonies of T7-PET pRSET A plasmid, so that we can select the successful ones next week.

The Wiki team made the outline of what our web page should look like, progress for them is really starting to take shape. Ethan also did research on a data related software called: Matlab

The HP team posted something about the "Aquatic Animal Day", 12 posts on total until now. Good work for them also.

We did have several T7-PET pRSETA plasmid bacterial candidate colonies

Today,(from the plates which we made last week) we will also need to do selections of pGal-PET plasmid. The process was very straight forward, we just picked out successful colonies from the plate like candidates and put them in a new one.

We also started the same thing we did last week for another gene: pGal-PET

The wiki team did the layout for their websites.

The HP team started gathering information about team members by making surveys, they gathered information like personalized profile and quotes.

We the wetlab team picked out pGal-PET candidate colonies from last week and once again made a lot of plates.

HP: Messaged teams in taiwan through instagram for collaborations:

• Survey: Are people interested in our final product, is the product going to be useful, household use possibilities
• Survey for experts: Landfill or industrial usage, price point at which the product could be sold, expert opinion on household use
• Contacting experts: in waste recycling, microplastics, government, groups working with plastic cleanup, etc.
• Collaborations: reaching out to Taiwanese teams, contacting slack chat HK group about the event they are planning to hold
• SDG experts: Iris Qui from last year
• Education: could do an event for the 7th graders this year during summer
• Science Fair: Use it as a survey opportunity to promote iGEM, get data from students on what they are interested in, and if we are to hold events in school, what they would like to do for tutorial sessions
• IHP: Put together a template of how we could introduce our project to specialists with possible questions, then we can edit it for specific people
• Proposal website: could hold an interview with the people, or collab for an instagram post

Mean While the wiki team starts filling out content for our web page.

Today we did plasmid extraction for triple mutation PETase. The concept of mutation in the protein's genomic sequence to directly affect the efficiency of an enzyme although isn't new to me, it is one of the thing that make me appreciate the extraordinary capacity of synthetic biology. If we really think about it, we are most likely currently living in the "early years" of synthetic biology.

HP group progress: Meeting notes:

• Present project (day 1 poster present)
• Day 2 is the actual seminar

• Focused on product and SDGs

• Short summary of the science fair and what we did for wiki page
• Instagram update on science fair

Group1: do bacterial transformation for IsT7-PETase ^{S121E/D186H/R280A} , and IspGal-PETase ^{S121E/D186H/R280A}

Group 2: set up 2X PCR for bacterial colonies candidates containing IsT7ase-PET ^{S121E/D186H/R280A} (set up #1-20)

Group 3: Do pGal1,10-IsPETase ^{S121E/D186H/R280A} plasmid extraction from bacterial colonies

All three experiments are consecutive so we did one group after another. I'm from group 1 so I did bacterial transformation, personally, I think bacterial transformation was the easiest protocol out of the three because there were much less step to cover.

For the HP group, They have sent out the "iGEM survey for teachers and students" and finished science fair posters.

Today we voted for what our team hoodie would look like, the two options were a sea otter icon and a badge with a whale, both of the accompanied with some plastic trash, the team settled with the sea otter icon.

Today we did western blot for all the protein products we have, in other words, all the mutations that successful.

Today (2024-8-24) is the first day of the TGem (Taiwan iGem) seminar the, the first day was just like any other typical "first days", we spent most of our times on introductions and experience sharing, regardless of the lengthiness of procedural events, we still managed to catch some time for our local teams. The next day was the day the teams presented their projects.

This is our team poster

Most of us are down to the final touch, some of us are working on our group presentation while others are doing functional assay.

Wiki: The wiki team are simplifying our wiki code to make them more accessible.

HP: The HP team is rather busy around this time, as for today, they:

• contact experts from difference fields.
• made surveys.
• organized spreadsheets and graphs of their surveyed data.

Wetlab: We did time course.

This week was the last chance to touch anything about the team, the finale.

Unsurprisingly, the wiki team worked on debugging the website and the wet lab worked on the final organizations of our current data, whilst making up for missing information. The debugging of the website was a lot tougher than we anticipated, it was almost as if the bugs were arbitrary. Bugs are discovered one after another.

Today we tried to find the physical docking site of PETase but to no avail, this was part of the personnel professor's request, however, the professor's request wasn't very specific so we had a hard time maneuvering the enzyme software, there's much to learn.

Today we finally had some progress with the docking, turns out, we should have applied our binding ligand with a PET polymer with three or more monomers, this checks out in almost every reference paper. This was such a whoopsy.

To make matters worse, we needed more time to generate an enzyme model with our current mutations. If we wanted to include this information, this step had to be complete because we could make sure whether the mutation would alter its conditions drastically or not.

Today the wet lab members did bacterial transformation, looking forward! Meanwhile the wiki team did more uploading and debugging almost finished the page.

wiki freeze!

Prepare for iGEM Jamboree

Prepare for iGEM Jamboree

iGEM Jamboree!!!