iGEM 2023 — Team lethbridgehs

{% block title %} Notebook {% endblock %}

{% block lead %} What did we do in the lab? {% endblock %}

![](https://static.igem.wiki/teams/4599/wiki/sky-bulb.png)

# Notebook

The lab book for this project can be found [here](https://static.igem.wiki/teams/4599/wiki/experiments/lab-book.pdf).

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© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/lethbridgehs](https://gitlab.igem.org/2023/lethbridgehs).

{% block title %} Engineering {% endblock %}

{% block lead %} Growth from Failure {% endblock %}

Engineering

Ideation

Re-Defining the Problem

Biosensor Design

Evaluation

Future Directions

![](https://static.igem.wiki/teams/4599/wiki/sky-bulb.png)

# Ideation

## ANA-Lyze This: Our Original Idea

During the initial planning stage, our team decided on a project that focused on the detection of Antinuclear Antibodies, a common indicator of many autoimmune diseases. With goals of providing accessibility, we envisioned â€śANA-Lyze thisâ€ť to be a biosensor based rapid test that could be used in the comfort of oneâ€™s home. Below is the â€śANA-Lyze Thisâ€ť logo created by team member, Tracy Wen.
![](https://static.igem.wiki/teams/4599/wiki/engineering/analogo.png)
Originally pitched by team members Steven Yang and Morteza Faraji, our team decided to take inspiration from the work done by Stewart Lohâ€™s lab. In their 2022 paper, they created a switchable GFP biosensor that could change color upon binding to a ligand. We planned to adapt these principles to chromoproteins, which would offer the advantage of easy visual detection without specialized equipment. Chromoproteins are unique in that they change color upon binding to a target molecule, making them suitable for a visual, user-friendly diagnostic test. We had grand ambitions for "ANA-Lyze This," envisioning it as a groundbreaking tool, similar to that of a Covid-19 test, that could be easily used by individuals at home.
![](https://static.igem.wiki/teams/4599/wiki/engineering/gfpdetectionmutant-fig2.png)
However, our journey took an unexpected turn when we presented our project idea to judges at the MindFuel's Tech Futures Challenge. Their feedback made us reconsider our project's direction. They pointed out several concerns, including the practicality of our project. It became evident that our initial project concept was not aligning with the desired global impact we sought.

![](https://static.igem.wiki/teams/4599/wiki/blue-problem.png)

# Re-Defining the Problem

In light of this feedback, our team decided to pivot away from the ANA detection sensor concept. We didn't abandon the innovative chromoprotein technology we had been exploring but instead refocused our efforts. We transformed our project into a versatile biosensor platform that could be adapted for various applications, Chromosense!
![](https://static.igem.wiki/teams/4599/wiki/engineering/img-7819.png)
Our newly devised project aimed to harness the color-changing properties of chromoproteins in a way that would be both impactful and practical. After careful consideration, we decided to address a different challenge: Velvet disease, a severe health issue in captive marine fish caused by Amyloodinium dinoflagellate parasites. The parasite infiltrates a fish's slime coat and damages its cells, posing a significant problem for fish farmers and pet owners.

![](https://static.igem.wiki/teams/4599/wiki/blue-dna.png)

# Biosensor Design

Using the constructs developed by the Uppsala iGEM team as our starting point, the focus of our biosensor is asPink and tsPurple, two remarkable chromoproteins that share the same chromophore sequence, CMYG. In order to create a switchable chromoprotein, the first step was to understand what makes asPink and tsPurple switch. What are the specific amino acids responsible for their distinct colors? Our journey takes us to amino acids I46, T94, and T95 in asPink. These particular amino acids drew our attention because they were not conserved between the two proteins and were predicted to interact with the chromophore in our structural models. Therefore, our strategy involves replacing these amino acids in asPink with their counterparts from tsPurple: T46, Q94, and I95. By doing so, we aim to pinpoint the essential amino acids that govern color change.
![](https://static.igem.wiki/teams/4599/wiki/engineering/aspinktspurple.png)
After site-directed mutagenesis, it was found that T94 and T95 were not essential for the colour differences. We have been unable to mutate I46 yet. Therefore, we need to go back to the amino acid sequences and investigate other differences. This will help identify how we can change asPink into tsPurple using strand displacement.

![](https://static.igem.wiki/teams/4599/wiki/blue-fish.png)

# Evaluation

![](https://static.igem.wiki/teams/4599/wiki/engineering/mermaid-diagram-2023-10-09-110541.png)
During the engineering cycle numerous challenges and unforeseen obstacles are encountered, underscoring the importance of project re-evaluation and adaptation of plans. Our experiences affirm that the initial phase, which involves defining the problem, is paramount and cannot be circumvented. A well-formed problem statement not only provides a clear direction but also serves as a goal throughout the development process. It has become evident that attempting to fit an existing technology to address a problem, without a thorough understanding and definition of the issue at hand, is a misaligned approach. Instead, the technology or solution developed should be tailored to meet the specific needs of the identified problem, ensuring that the following stages of the engineering cycleâ€” from conceptualization to implementationâ€” are effectively aligned toward a viable solution.

![](https://static.igem.wiki/teams/4599/wiki/blue-glass.png)

# Future Directions

As discussed on our [Project Description](https://2023.igem.wiki/lethbridgehs/description#option_5_header) page, many steps are necessary to produce a working biosensor, which we can implement to further our project.

![](https://static.igem.wiki/teams/4599/wiki/blue-scroll-top.png)

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Â© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/lethbridgehs](https://gitlab.igem.org/2023/lethbridgehs).

{% block title %} Experiments {% endblock %}

{% block lead %} How to make ChromoSense {% endblock %}

Experiments

Workflow

Design

Methods

![](https://static.igem.wiki/teams/4599/wiki/sky-bulb.png)

# Workflow

![](https://static.igem.wiki/teams/4599/wiki/experiments/flowchart.png)
Our team's goal in the lab was to complete site-directed mutagenesis on the pSB1C3-asPink plasmid to elucidate essential amino acids for the colour.

![](https://static.igem.wiki/teams/4599/wiki/blue-problem.png)

# Design

Mutagenic primers were designed to change T94 and T95 in asPink to Q94 and I95. A Hpy188III site was removed using a silent mutation to change the restriction pattern of the mutant plamsid.

fwd primer
5' - GTACCCAGATTTATGAAGATGGCGGTTTTCTGACG - 3'
rev primer
5' - CATAAATCTGGGTACGTTCCCAGGTAAAGCC - 3'
![](https://static.igem.wiki/teams/4599/wiki/experiments/aspinkmapoct2023.png)

1. Mutagenic primers are used during PCR to introduce point mutations to the new plasmid.
2. The PCR reaction is treated with DpnI to digest the orignal non-mutant plasmid.
3. The PCR product is transformed into *E. coli* and grown on selective media.

![](https://static.igem.wiki/teams/4599/wiki/experiments/sdmflow.png)

![](https://static.igem.wiki/teams/4599/wiki/blue-dna.png)

# Methods

All protocols can be found [here](https://static.igem.wiki/teams/4599/wiki/experiments/protocols.pdf).

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© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/lethbridgehs](https://gitlab.igem.org/2023/lethbridgehs).

{% block title %} Results {% endblock %}

{% block lead %} What happened in the lab? {% endblock %}

Results

DNA Isolation

SDM

Restriction and Sequencing

Observations

Future Directions

![](https://static.igem.wiki/teams/4599/wiki/sky-bulb.png)

# DNA Isolation

Plasmids were ordered from Addgene.org and grown in LB broth. Both tsPurple and asPink are constituatively expressed. Cells were collected and the plasmids were isolated using the BioBasic DNA isolation kit.
![](https://static.igem.wiki/teams/4599/wiki/results/flasksgrowth.jpg)
The DNA was run on a 1% agarose gel and then stored at -20C for future use.

![](https://static.igem.wiki/teams/4599/wiki/blue-problem.png)

# Site-Directed Mutagenesis

Several attempts were made using the mutagenic primers and the asPink plasmid to produce new mutated plasmid with the appropriate changes.
![](https://static.igem.wiki/teams/4599/wiki/results/sdmgel.png)
Using the Q5 DNA polymerase, we were successful in completing the SDM reaction to produce the new pSB1C3-asPinkTT9495QI.

![](https://static.igem.wiki/teams/4599/wiki/blue-dna.png)

# Restriction Digestion and Sequencing

![](https://static.igem.wiki/teams/4599/wiki/results/hpydigestion.png)
Transformation into DH5alpha resulted in several pink colonies on selective media. Individual colonies were selected for DNA isolation, Hyp188III digestion, and sequencing. It was found that all colonies selected had the correct digestion pattern. When the DNA was sent for [sequencing](https://static.igem.wiki/teams/4599/wiki/results/results-clustal-omega-multiple-sequence-alignment-embl-ebi-3.pdf) the correct DNA changes were found.
![](https://static.igem.wiki/teams/4599/wiki/results/img-7461-1.jpg)

![](https://static.igem.wiki/teams/4599/wiki/blue-fish.png)

# Observations

We were able to change T94 and T95 in asPink to the corresponding amino acids Q94 and I95 of tsPurple. The change did not result in a colour shift, as the mutant protein was still pink, but it did result in a much slower maturation of the protein. Colour was only observed in the bacterial colonies after more than 24 hours and a period of incubation at 4 degrees C.

![](https://static.igem.wiki/teams/4599/wiki/blue-glass.png)

# Future Directions

The next steps in the lab will mean designing additional mutagenis primers to change any other identified amino acids to those in tsPurple. This will be a long process.

The asPink gene should also be placed in a plasmid that has a purification tag. This would allow us to purify and test the protein in the future.
Engineering of *P. denitrificans* should begin. First, to test if the chromoprotein can be expressed in the bacteria and the colour observed. Second, to test how the engineered strain competes against the other naturally occuring bacteria in the filtration system. Finally, to see if this bacteria is able to detect any pathogens in the aquatic system. More info on biosafety measured can be found [here](https://2023.igem.wiki/lethbridgehs/safety).

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© 2023 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2023/lethbridgehs](https://gitlab.igem.org/2023/lethbridgehs).