iGEM 2024 — Team washington

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## November

#### Week 4

* November 26, 2023: First iGEM meeting of this season! Past UW iGEM members talked about the expectation of this season and started to develop project ideas.

## December

#### Week 1

* During week 1 members continued to work on detailing their individual project ideas and created proposals for them.

#### Week 2-3

* After two weeks of background research the five topics that were developed into a proposal are continuing last year’s project, using kelp to combat ocean acidification, pharmacogenetics, enzymatic blood type conversion, and gut microbiome metabolite.
* December 20, 2023: Team members presented their ideas and a vote was casted. The project idea that received the highest amount of votes is pharmacogenetics, in which we could increase expression of certain genes to increase or decrease an individual’s ability to metabolize medicines.

#### Week 4

* Started the onboarding process for the new team members.

## January

#### Week 4

* January 28, 2024: First official meeting with the new iGEM team members this year! Introduced the basics of iGEM to the new members.
* Project ideas were introduced to the new members, subteams continued background research to refine the project idea.

## February

#### Week 1

* Based on our initial project idea of pharmacogenetics we started discussing the details of the project that we would like to do this year.
* Started background research regarding the type of cancers that is more likely to be affected by pharmacogenetics.

#### Week 2

* Discovered that there might be novelty issues in our current ideas.
* Continued literature review in regards to the types of cancer that we looked into.

#### Week 3 to 4

* Individual members were assigned different topics they were interested in and continued literature review.

## March

#### Week 1

* March 3, 2024: Abacavir, Irinotecan, CYP3A, and OCT1 were proposed as the four possible directions for our project idea.

#### Week 2

* Further research was done regarding the UGT1A1 disease by the human practices subteam.

#### Week 3 to 4

* Discovered novelty issues regarding the current proposed ideas, continued literature review and background research.

## April

#### Week 1

* Discovered that neutropenia and antibiotics resistance are some of the more pressing issue that cancer patients are facing everyday.

#### Week 2

* Wetlab decided that our project will be on how we will use human albumins to extend the half life of antibiotics that are essential to cancer patients.

#### Week 3

* Wetlab met with their mentor Dr. Jennifer Kong and discussed about the details of our experiments.
* Wetlab members started safely trainings and talked about the general timeline.

#### Week 4

* April 28, 2024: Confirmed with the entire subteam that our project idea will be using human serum albumin to extend the half-life of cephalosporins by binding them together.
* After confirmation of the project, wetlab started researching about what is an appropriate albumin binding moieties as a base model that we can modify.

## May

#### Week 1

* It is decided that we will create an albumin-drug complex through the binding of human serum albumin with a modified ABM, a linker, and a cephalosporin.
* Wetlab started researching possible ways to quantify the binding between the ABM and human albumin.
* Looked into which type of antibiotics is most relevant for cancer patients.

#### Week 2

* Wetlab looked for a way to quantify peptide binding and a way to quantify free flowing drugs and looked into how to differentiate bound albumin from unbound albumin.

#### Week 3

* Wetlab decided that we will employ a 2-step-verification process. We will first isolate the unbound albumins then find quantification it possibly using liquid photometry or GC-MS.

#### Week 4

* Wetlab started and finished the Safety form required for iGEM registration.
* Drylab is analyzing and modifying the ABM to increase its affinity to human serum albumin.

## June

#### Week 1

* Written protocols for the expression and purification of our modified ABM protein.

#### Week 2

* Wetlab looked into ways that we can quantify the binding of albumin with ABM and ABM with linker.
* Researched more about the properties of cephalosporins and compared the third generation with the fourth generation.
* Through interviews with experts, human practices found out that fourth generation cephalosporins is the most common antibiotic used in cancer patients currently.

#### Week 3

* Wetlab looked into ways to synthesize the linker maleimide-PEG-OH and created protocols.
* Looked into ways for quantification of the linker possibly through making the linker fluorescent.

#### Week 4

* Contacted professors in the Chemistry department of University of Washington to seek guidance on performing the chemistry to synthesize linkers.

## July

#### Week 1

* Wetlab started designing the plasmid necessary for the AMB protein expression.

#### Week 2

* Refined and completed our protocols for protein synthesis and protein purification.
* Started the thiol reaction and esterification protocols for linker synthesis.
* Contacted staff in the chemistry department to inquire about the use of NMR and GC-MS machines for future quantification processes.

#### Week 3

* Researched about the use of equilibrium dialysis to separate the unbound albumin from the bound albumin.
* Developed specific protocols for separation of proteins with similar size using equilibrium dialysis.

#### Week 4

* Received feedback from Fernando Mejia regarding the protocols that wrote, and started refining the protocols according to his advice.
* Placed orders for the necessary materials that we need to perform the experiment.

## August

#### Week 1

* Contact people in the Biochem core regarding equipment use and training for size exclusion chromatography and HPLC.
* Went over the use of Benchling and proper lab notebook recording format.

#### Week 2 to 4

* While waiting for the supplies to arrive, wetlab focused on contacting people to inquire uses and price of specific equipment in the meantime.

## September

#### Week 1 to 3

* While waiting for the supplies to arrive, wetlab started researching for opportunities to fundraise for the team.

#### Week 4

* September 27, 2024: The plasmids for our modified ABM protein finally arrived.
* September 28, 2024: With the help of Dr. Jennifer Kong, wetlab started the plasmid prep.
* September 30, 2024: With the help of Dr. Jennifer Kong, wetlab started protein extraction and performed western blot.

Please view this wiki on a computer for the best experience!

# Washington iGEM

2024 Project MightyMoieties

uwigem@uw.edu

## General

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

## Dry Lab

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

## Wet Lab

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## Social Media

[![](https://static.igem.wiki/teams/5126/facebook-f-brands-solid.svg)](https://www.facebook.com/WashingtoniGEM)
[![](https://static.igem.wiki/teams/5126/instagram-brands-solid.svg)](https://www.instagram.com/washington_igem/)
[![](https://static.igem.wiki/teams/5126/x-twitter-brands-solid.svg)](https://twitter.com/uw_igem_team)
[![](https://static.igem.wiki/teams/5126/github-brands-solid.svg)](https://github.com/uwigem)
[![](https://static.igem.wiki/teams/5126/youtube-brands-solid.svg)](https://www.youtube.com/%40WashingtoniGEM)

© 2024 - 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/2024/washington](https://gitlab.igem.org/2024/washington).

# NOTEBOOK

![image](https://static.igem.wiki/teams/5126/blackcirclev2-removebg-preview.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}
Innovative educational tools and outreach activities have the ability to establish a two-way dialogue with new communities by discussing public values and the science behind synthetic biology.
{% endblock %}
{% block page\_content %}

## Overview

Driven by the need to improve how cancer patients retain antibiotics in their system, our team developed a novel method involving a human albumin linking process to extend the half-life of these medications. Our project is guided by the principles of the engineering cycle. At a macro level, our approach follows an iterative design-build-test-learn process, transitioning from dry lab to wet lab. Additionally, our work is refined through micro iterations of the engineering cycle, focusing on the finer details to ensure optimal results.

Please **click** on the **four components of the engineering design cycle graphics on the left** to see all parts of our cycle iterations!

# Protein Modeling

## Albumin Binding Moiety Computational Design

Learn

Design

Build

Test

Learn about our programs and opportunities.

# Wetlab (Micro)

## Albumin-ABM Binding Design Cycle 1: Protein Synthesis

Learn

Design

Build

Test

Learn about our programs and opportunities.

## Cycle 2: Induced Binding

Learn

Design

Build

Test

Learn about our programs and opportunities.

## Albumin-ABM-Linker Binding Design Cycle 1

Learn

Design

Build

Test

Learn about our programs and opportunities.

## Albumin-ABM-Linker-Cefepime Binding Design Cycle 1

Learn

Design

Build

Test

Learn about our programs and opportunities.

Please view this wiki on a computer for the best experience!

# Washington iGEM

2024 Project MightyMoieties

uwigem@uw.edu

## General

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

## Dry Lab

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

## Wet Lab

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## Social Media

[![](https://static.igem.wiki/teams/5126/facebook-f-brands-solid.svg)](https://www.facebook.com/WashingtoniGEM)
[![](https://static.igem.wiki/teams/5126/instagram-brands-solid.svg)](https://www.instagram.com/washington_igem/)
[![](https://static.igem.wiki/teams/5126/x-twitter-brands-solid.svg)](https://twitter.com/uw_igem_team)
[![](https://static.igem.wiki/teams/5126/github-brands-solid.svg)](https://github.com/uwigem)
[![](https://static.igem.wiki/teams/5126/youtube-brands-solid.svg)](https://www.youtube.com/%40WashingtoniGEM)

© 2024 - 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/2024/washington](https://gitlab.igem.org/2024/washington).

# ENGINEERING

![image](https://static.igem.wiki/teams/5126/blackcirclev2-removebg-preview.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Please refer to our protocol section for detailed steps of our experiments.

#### Experimental Design

Our experiment consists of three main sections, synthesizing the modified protein, induce the binding of different components, and testing the affinity of our ABM design.

#### Protein Synthesis

Starting with protein synthesis, we will first express the plasmid we designed in E. coli. We have streaked the bacteria on glycerol plates and will induce protein expression using IPTG. Following induction, we will harvest the cells and purify the expressed protein through affinity chromatography. To quantify the purified protein, we will perform a Western blot analysis, allowing us to confirm the expression and assess the yield of our target protein.

#### Induce Binding

Following the synthesis of our modified ABM, we will proceed to combine all components of our target complex.

First, we will induce the binding between albumin and ABM by placing them in the same environment. Next, we will synthesize the linker maleimide-PEG-OH through a thiol reaction, followed by its binding with Cefepime via esterification. The final product will be an albumin-ABM-linker-Cefepime complex, which can then be purified and prepared for administration to patients.

#### Binding Evaluation

Throughout each step of the binding process, we will implement a quantification strategy to confirm the success of our binding experiment. This will involve a two-step verification process: isolation followed by quantification.

We will utilize equilibrium dialysis to separate the bound products from the unbound materials. Subsequently, we will employ GC-MS to quantify the amount of bound products, allowing us to infer their affinity.

#### Discussion

Due to complications with plasmid design and shipping processes, we were unable to progress beyond the protein synthesis stage. If our design proves successful, we anticipate that cefepime will effectively attach to human serum albumin, and our modified ABM will enhance the overall affinity of the complex. We expect that cefepime will be retained in the bloodstream for extended periods, thereby reducing the frequency of drug administration and improving patients' quality of life.

#### Protocols

[Transformation](https://static.igem.wiki/teams/5126/igem24-transformation-protocol.pdf)
[Purification](https://static.igem.wiki/teams/5126/igem24-purification-protocol.pdf)

Please view this wiki on a computer for the best experience!

# Washington iGEM

2024 Project MightyMoieties

uwigem@uw.edu

## General

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

## Dry Lab

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

## Wet Lab

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## Social Media

[![](https://static.igem.wiki/teams/5126/facebook-f-brands-solid.svg)](https://www.facebook.com/WashingtoniGEM)
[![](https://static.igem.wiki/teams/5126/instagram-brands-solid.svg)](https://www.instagram.com/washington_igem/)
[![](https://static.igem.wiki/teams/5126/x-twitter-brands-solid.svg)](https://twitter.com/uw_igem_team)
[![](https://static.igem.wiki/teams/5126/github-brands-solid.svg)](https://github.com/uwigem)
[![](https://static.igem.wiki/teams/5126/youtube-brands-solid.svg)](https://www.youtube.com/%40WashingtoniGEM)

© 2024 - 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/2024/washington](https://gitlab.igem.org/2024/washington).

# EXPERIMENTS

![image](https://static.igem.wiki/teams/5126/blackcirclev2-removebg-preview.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# RESULTS

![image](https://static.igem.wiki/teams/5126/blackcirclev2-removebg-preview.webp)

#### Computational Binder Design

Our protein modeling team designed binders using machine learning-based tools, as described on our [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page. The sequence results for novel albumin binding moieties are attached below:

## designed\_sequences.fasta ▶

> 1512K44C|DIFFUSION NOVEL STRUCTURE LENGTH 53

MSRKKERAEELYNTALLSARRGNKKAAERAAEIILEDTGDEEAACKAREALKAI

> 2630E2C|PARTIAL DIFFUSION STRUCTURE LENGTH 53

MSCEEKKIEEHKKKILAELDALGINNKLIKAEIRKSKIPEDMETLFEEIKAERA

> 1712E8C|DIFFUSION NOVEL STRUCTURE LENGTH 40

MDLLKKADCKAKEANELQRKGGKLSDIMKLVKEAEELREAA

#### Laboratory Results

![Description of Image](https://static.igem.wiki/teams/5126/results-img.jpg)

Figure 1. Purified ABD035\_C22, ABD035\_C29, ND54\_C45, and PD54\_C3 protein
Gel showing the presence of our modified protein.

Due to the difficulty of procuring plasmids in time for our project, we were unable to test the binding affinity of our modified albumin binding moieties (ABM). If we were to test the binding affinity we would measure the amount of ABM that successfully bound to human serum albumin through equilibrium dialysis and compare the binding percentage between the different modified ABM and our wild-type ABM.

#### Next Steps

Once we confirm that our modified ABM exhibits improved affinity for human serum albumin, our next step will be to connect this complex with a fourth-generation cephalosporin. We plan to begin by synthesizing a linker capable of facilitating click chemistry. Using Cefepime as a representative fourth-generation cephalosporin, we will then bind the antibiotic to the albumin-ABM complex and measure whether the antibiotic is successfully incorporated into the complex.

Please view this wiki on a computer for the best experience!

# Washington iGEM

2024 Project MightyMoieties

uwigem@uw.edu

## General

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

## Dry Lab

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

## Wet Lab

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## Social Media

[![](https://static.igem.wiki/teams/5126/facebook-f-brands-solid.svg)](https://www.facebook.com/WashingtoniGEM)
[![](https://static.igem.wiki/teams/5126/instagram-brands-solid.svg)](https://www.instagram.com/washington_igem/)
[![](https://static.igem.wiki/teams/5126/x-twitter-brands-solid.svg)](https://twitter.com/uw_igem_team)
[![](https://static.igem.wiki/teams/5126/github-brands-solid.svg)](https://github.com/uwigem)
[![](https://static.igem.wiki/teams/5126/youtube-brands-solid.svg)](https://www.youtube.com/%40WashingtoniGEM)

© 2024 - 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/2024/washington](https://gitlab.igem.org/2024/washington).

{% endblock %}