iGEM 2022 — Team gastonday-shangde

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{% block title %}Notebook{% endblock %}
{% block lead %}We kept track on our lab process and here is our Lab Notebook!{% endblock %}
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#### Lab Noatebook Pictures

Links to Lab Notebook Pictures

Open a PDF file [Lab Notebook](https://static.igem.wiki/teams/4481/wiki/sharpdevice-gastonday-org-20221011-092224.pdf).

Open a PDF file [Lab Notebook](https://static.igem.wiki/teams/4481/wiki/1234.pdf).

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Here is our engineering cycle!!{% endblock %}
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#### Our Engineering Design Cycle

Design: The literature provides sequence but it contains illegal restriction sites so the sequence is modified to remove the sites and then optimized for use in *E. coli*.

Build:The part was built by IDT as a plasmid and then transformed into *E. coli*.

Test: We ligated the coding sequence to a stop sequence and successfully transformed into *E. coli*. We then ligated that to a strong constitutive promoter but the transformations failed multiple times.

Learn: Additional research showed that the intermediates in this pathway can be used as antimicrobials in some systems. Constitutive expression may be killing the transformed cells.

Redesign: Pull low level constitutive promoters, arabinose-inducible promoters, and osmotic pressure-inducible promoters to retry. This is currently in progress.

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{% block title %}Experiments{% endblock %}
{% block lead %}Starting with deep research and reading papers, we disvered several ideas about helping the patients.{% endblock %}
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We first did paper research, learned about the basic knowledge about Nonketotic Hyperglycinemia, and brainstormed about how we can develop a design to help the patients. In a particular paper, we found a method of carrying out the phenylalanine-to-cinnamaldehyde metabolic pathway in modified *E. coli* and learned the method. We bought particular genes including SmPAL, ScCCL, and AtCCR from IDT and added them into *E. coli* cells that we have.

In this process, we used the following materials:

- *E. coli* K-12
- Standard bacterial growth media and equipment
- DNA Ligation Kit (New England Biolabs)
- Plasmid Isolation Kit (New England Biolabs)
- Restriction enzymes (New England Biolabs)
- Agarose
- Gel electrophoresis buffer
- SDS-PAGE Premade Gels (Bio-Rad)

\*note that the bacteria used in the experiments are under biosafety level 1

We went through digestion to cut the DNA in the *E. coli*. We went through ligation and transformation to link the added gene with *E. coli*’s original gene. To test if we successfully added the gene into the *E. coli* cells, we first picked colonies, and then we went through plasmid preparation and PCR confirmation to get ready for gel electrophoresis. After we observed the size of the DNA through gel electrophoresis and ensured that the genes were added into the *E. coli* cells, we went through cryopreservation to store some of our edited *E. coli* away. Later, we worked on adding the stop codon onto the gene we added into *E. coli*, and went through the cycle of digestion, ligation, transformation, pick colonies, plasmid preparation, PCR confirmation, and gel electrophoresis several times.

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{% block title %}Results{% endblock %}
{% block lead %}Here are our project results!{% endblock %}
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#### Our Results

The team successfully added SmPAL, ScCCL, and AtCCR into *E. coli* cells. All three gene sequences were codon optimized for use in *E. coli*. Also, we successfully added the stop codon on the three genes in the *E. coli* cells. This progress paved the way for us to express the genes in the *E. coli* cells and produce the enzymes that work in the phenylalanine-to-cinnamaldehyde pathway. Our team has put the parts into the registry and had a written procedure on the experiment page of our wiki so that if people want to replicate the experiment, they have the information to do so.

We have been working on adding the promoters to the genes we previously added into the *E. coli* cells. We went through the experimental cycle, including digestion, ligation, transformation, pick colonies, plasmid preparation, PCR confirmation, and gel electrophoresis several times and such processes require a lot of time investment. Due to limitations in funding and equipment, we do not have the kits that can allow the process to go faster. We have tried identifying the problems and adjusting our procedures and experimental design. Still, it is pretty frustrating that we are not quite seeing things working as we are expecting. We are, and we will continue working on the wet lab part of our project, carrying great hope to see some progress.

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