iGEM 2023 — Team rbhs-sandiego-ca

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{% block title %}Notebook{% endblock %}
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## Lab Notebook

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Below we have listed all our lab notes and meeting notes that we have taken this whole year.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}one pawsitive blueprint at a time{% endblock %}
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## First Iteration of the Engineering Cycle

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#### Research and Design

Our project primarily functions based on being able to use the functional epitopes of the tyrosine-rich oocyst wall protein (TrOWP2) derived from Toxoplasma Gondii to produce an immunological response from the cat, thus preventing future toxoplasmosis and reducing shedding of the parasite into the environment. The peptides that comprise linear B-cell epitopes can be used in place of antigens when producing antibody responses via immunization. As such, we can attach an epitope peptide sequence to our phage-based vaccine in order to effectively immunize the cat. To better understand and determine the locations of these epitopes, we used several web-based software in order to predict, as well as visualize the location of the antigen’s epitope region. We also found other antigen sequences through literature review and selected the two antigens with the highest levels of antigenicity.

Using the newfound sequence FKCAEGTTETIDGDCKRLKQFPP, Alpha Fold via the collabfold web server was used to produce 3D protein models that we could then use for additional analysis of the epitope’s properties based on the structure. With these models, we predicted the epitope sequence to be TTTAAATGTGCCGAAGGAACAACGGAGACTATTGACGGTGACTGCAAGCGCTTGAAGCAGTTTCCGCCA. This became our part BBa\_K4734005. The other two epitope sequences were TNNEDEQ (which became our part BBa\_K4734003) and QGNDEHSSQ (which became our part BBa\_K4734004). We found the DNA sequences of these through literature review.

We selected the phage M13 to perform our peptide display with; we needed to design primers to amplify the M13 vector. We used Benchling to design two sets of primers. We made one of our sets of primers into parts. Our forward primer from that set became the part BBa\_K4734001 and our reverse primer from that set became the part BBa\_K4734002.

#### Build

We performed a PCR with our template DNA with each set of primers we designed in order to linearize our vector, so that we may insert our epitope into the vector. This leads to the M13KE Phage being able to present the phage and the epitope attached to it so that it can penetrate through the mucus layer and epithelial cells to the lymph in order to produce an immune response. Then, we performed gel electrophoresis on each sample to determine if we had DNA of the correct size. After confirming our vector was around 7 kb as expected, we annealed our 3 antigen insert sequences and ligated them to our vector in separate samples.

#### Test

We did a gel test on the ligated pieces in order to see if the piece was ligated properly. We found no bands on our DNA gel, confirming there was an error in our ligation step.

![](https://static.igem.wiki/teams/4734/wiki/img-7114.jpeg)

#### Learn

We rationalized that the lack of band presence was due to a mistake in our ligation or plasmid linearizing step. We recognized some issues with our PCR protocol, which we discuss further in the next section. We also note that the frequent inaccuracies in blunt-end ligation could have also been a key factor in the failure of our ligation.

## Second Iteration of the Engineering Cycle

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#### Research and Design

We used Benchling to design our primers. We chose sequences on either side of the site of insertion that were an agreeable length, between about 20 and 30 base pairs, and had matching annealing temperatures. We created two sets of primers.

![](https://static.igem.wiki/teams/4734/wiki/screen-shot-2023-10-12-at-7-13-16-am.png)

#### Build

We ordered our sequences with Integrated DNA Technologies (IDT) in order to begin testing. We resuspended our primer upon arrival, and performed a 1:10 dilution on each primer so that we may use them for the PCR amplification protocol. We then amplified the linearized plasmid because the primers were placed in a manner that would cause them to create a kink in the middle of the M13KE vector. We followed the protocol listed below, except for once change in which we used the Klenow Polymerase Fragment:

![](https://static.igem.wiki/teams/4734/wiki/screen-shot-2023-10-12-at-7-09-45-am.png)

#### Test

In order to verify the length and correct linearization of the vector, we conducted a 1% DNA gel in order to test our size. We ran our entire volume on the gel, which was 20 microliters. According to our gel below our sequences matched up with the same length that we expected; therefore, we concluded that the linearization was accurate. The expected size was around 7000 bp.

![](https://static.igem.wiki/teams/4734/wiki/screen-shot-2023-10-12-at-7-11-58-am.png)

#### Learn

Our sequences matched up, so we learned that these primers ere the correct ones that we designed properly in order to effectively linearize the vector, so that we may insert our epitope onto the phage vector.

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{% block title %}Experiments{% endblock %}
{% block lead %}our cat-alyst for discovery{% endblock %}
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## Overview

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All lab work completed was done at the San Diego State University under the supervision of Dr. Forest Rohwer, Nelson Vyada, and Vanessa Salcido. The following page includes all experiments conducted in lab as well as a brief overview of their purposes

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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