iGEM 2023 — Team ohio-state-fabe

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April

## Wet Lab

#### 04/12/2023

- Completed Shark Tank style presentations to the group
- Decided to pursue a Type 3 Secretion System Project
- Began to set out timeline for future of project with a Gantt chart

#### 04/19/2023

- Brainstormed protein disorders to target
- Defined Project Version 1: Some diseases cause incorrect protein folding. We will use T3SS to replace these proteins or their function

## HP

#### 4/20/2023

- Emailed Dr. Ruano-Gallego and Dr. Luis Ángel Fernández from the Spanish Research Council (CSIC) about their success in expressing Type III Secretion Systems in *Escherichia Coli*  K-12
- Were instead referred to Dr. Cammie Lesser from Harvard University

May

## Wet Lab

#### 05/11/2023

- Meeting with Dr. Lesser from Harvard University
- Discussed conceptualization for our project and collaboration, arranged MTA

#### 5/19/2023

- Meeting with Dr. Immethun who is a synthetic biologist from Battelle
- Received feedback on presentation of our project and the direction/scale of the idea
- Researched potential therapeutic proteins/nanobodies to target with delivery system, reached out to authors for more discussion
- Began brainstorming protocols

## Dry Lab

#### 5/3/2023

- Began research on a support vector machine (SVM) model to identify proteins that could secrete through T3SS and successfully refold
- Identified DeepT3 as a project of interest for protein identification

#### 5/10/2023

- Realized DeepT3 would not work because it was tuned for effector protein identification from Shigella genome
- Created list of attributes we could use to fingerprint different proteins of interest

#### 5/17/2023

- After meeting with Dr. Lesser, the team pivoted to nanobody research, and scrapped the SVM idea because nanobodies often fold successfully

## HP

#### 5/5/2023

- Explored potential collaboration with other iGEM teams in Ohio, including Wright State University’s (WSU) team

#### 5/19/2023

- Presented the initial Type III Secretion System project to science and technology company, Battelle, to practice our speaking skills and get technical opinions on our project

#### 5/20/2023

- Released information about our 2023 project on our Instagram

#### 5/28/2023

- Researched other student organizations affiliated with Ohio State to collaborate with them to promote synthetic biology outreach

#### 5/31/2023

- Explored options for experiments to present to The Center for Science and Industry (COSI) in the upcoming meeting

June

## Wet Lab

#### 6/15/2023

- Asked Dr. Lesser about specific questions and feasibility

#### 6/20/2023

- Response from Dr. Lesser which included the sequences for N-terminal secretion sequences
- Email included a brief description of Dr.Lesser’s research and gave advice on how to go about the rest of the experiment such as how to start

## Dry Lab

#### 6/14/2023

- Looked at previous iGem teams to get an idea of what we wanted to do, began work on infographic

#### 6/21/2023

- Kickstarter python training with download tutorial, began to think about what professionals to contact

#### 6/28/2023

- Found several dry lab professionals to contact, made questionnaire sheet for professionals, python tutorial pandas library

## HP

#### 6/2/2023

- Zoomed with COSI alongside WSU to plan a date to visit the museum to teach about synthetic biology and present thorough descriptions of our respective 2023 projects

#### 6/7/2023

- Met via zoom to discuss experiment ideas for COSI

#### 6/17/2023

- Further brainstormed ideas for COSI and general setup

#### 6/21/2023

- Met via zoom to discuss additional ways to educate the local community about synthetic biology and type III secretion systems. Explored using 3D models and collaborating with local Girl Scout troops in need of their “Think like an Engineer” journey

July

## Dry Lab

#### 7/5/2023

- Went over python assignment to teach how to analyze and graph data in python (libraries, syntax, spacing)

#### 7/12/2023

- Biocad discussion & elected team member to research it, reached out to set up meeting with Lindsey (Graduate Student Advisor)

## HP

#### 7/2/2023

- Posted an extensive description on the contents of the 2023 project on our Instagram
![HP1](https://static.igem.wiki/teams/5027/wiki/hp1.png)
![HP2](https://static.igem.wiki/teams/5027/wiki/hp2.png)
![HP3](https://static.igem.wiki/teams/5027/wiki/hp3.png)

#### 7/11/2023

- Met to discuss availability for COSI and the progress made on models and experiments to be presented there

#### 7/21/2023

- Finalized experiments to be conducted at COSI and developed procedures for each

August

## Wet Lab

#### 8/8/2023

- Conducted lab training videos where Satvik (Graduate Student Wet Lab Advisor) went over how to use each of the devices

#### 8/9/2023

- Requested information regarding the plasmid maps for the Flag-tagged Shigella Effector and nanobody that were used as positive controls by Dr. Lesser’s lab

## Dry Lab

#### 8/2/2023

- Questioned Jay (Wet Lab Lead) for wet lab information, decided to do a modeling approach, planned to meet with a professional Dr. Ramnath for machine learning, added questions to professional questionnaire

#### 8/28/2023

- Discussed professionals we have emailed and how they have helped our project
- Elijah updated the team on his modeling work

## HP

#### 8/3/2023

- Extensively detailed remaining tasks and distributed the work equitably

#### 8/14/2023

- Collaborated with the Wright State University iGEM team to teach synthetic biology and about our respective projects at the Boonshoft Museum of Discovery
![Boonshoft](https://static.igem.wiki/teams/5027/wiki/boonshoft.jpg)

#### 8/15/2023

- Posted Instagram stories about our trip to Boonshoft to teach our followers about synthetic biology

#### 8/27/2023

- Posted three patient surveys into a broad spectrum of subreddits of patient subgroups affected with gastrointestinal disorders to obtain feedback on willingness to use T3SS therapies

September

## Wet Lab

#### 9/5/2023

- The team was informed that the laboratory would not be available until the end of October 2023

## Dry Lab

#### 9/13/2023

- Started prioritizing the wiki and continuing to work on it
- Helped Elijah (Team Co-President) with the modeling
- Wiki tutorials/Wiki101

#### 9/28/2023

- Continued to work on wiki, got video uploaded, pictures and home page done
- Finished wiki writeups needed for Drylab, learning HTML as a team

## HP

#### 9/2/2023

- Volunteered at COSI with the Wright State University iGEM team to bring synthetic biology to young students

![COSI](https://static.igem.wiki/teams/5027/wiki/cosi.jpg)
![COSI2](https://static.igem.wiki/teams/5027/wiki/cosi2.jpg)
![COSI5](https://static.igem.wiki/teams/5027/wiki/cosi5.jpg)

#### 9/3/2023

- Included photos from COSI on our Instagram story to show our followers how they can perform easy scientific experiments at their home

![COSI3](https://static.igem.wiki/teams/5027/wiki/cosi3.jpg)
![COSI4](https://static.igem.wiki/teams/5027/wiki/cosi4.jpg)

#### 9/30/2023

- Team attended a science and sustainability event, WestFest, to teach others about synthetic biology and our project
- Took many pictures to post on our social media

![WF1](https://static.igem.wiki/teams/5027/wiki/wf1.jpg)
![WF2](https://static.igem.wiki/teams/5027/wiki/wf2.jpg)
![WF3](https://static.igem.wiki/teams/5027/wiki/wf3.jpg)

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#### Engineering Therapeutic Applications for Type 3 Secretion Systems Using Nanobodies

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#### Find our wet lab experiments in the below pdf! They can also be downloaded [here.](https://static.igem.wiki/teams/5027/wiki/experiments.pdf)

The initial plan was to work in a lab, so this is what we would have if our lab was ready in time.

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#### **Project Achievements**

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#### Dry Lab

This year, our dry-lab was able to successfully model the lead sequence optimization process for secretion via T3SS in Java. We implemented and considered many biological functions in our code and assigned biological activity values for each output sequence, which allowed us to visualize how the system was optimizing the sequence across generations. Building and studying this model gave us insight into the function of T3SS and prepared us to design sequences that would efficiently secrete.

The current program takes a sequence of 20 nucleotides and optimizes it to obtain the highest possible activity value. This is useful for optimizing the lead sequence as it allows for lead sequence to achieve a higher biological activity rate. By doing so, one could splice together the optimized lead sequence and another strand in order to help a protein or other substance proliferate.

In the below graph, the optimized sequence for each generation is generated and then the optimized biological activity value for each generation is then also generated. The average activity column is included due to the fact that the program starts off with a random population value and then mimics the spreading of that population and also somewhat mimics crossover and natural selection as well. These processes were added in order to mimic what may go into optimizing a lead sequence. Due to the structure of the code, at some point, the code fails to optimize the code further then. At 0.99 biological activity, the program can not properly calculate relative to a value close to 1 so it begins to spit values that are not optimized.

![WF1](https://static.igem.wiki/teams/5027/wiki/model2.png)
![WF1](https://static.igem.wiki/teams/5027/wiki/model1.jpg)

Before deciding on our DiGI-T3 lead-sequence optimizer, we had other ideas for modeling some part of the therapeutic T3SS design cycle. The first idea that the team wanted to pursue was a support vector machine (SVM) model to determine a statistical probability of secretion based on protein fingerprinting criteria. However, our meeting with Dr. Lesser taught us that secretion does not guarantee proper folding, and that this folding is more often the issue. Her recommendation was to focus instead on nanobodies, which almost always fold correctly due to their tiny size.
![WF1](https://static.igem.wiki/teams/5027/wiki/model4.png)

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#### Human Practices

For human practices, our team was involved in many outreach events and discussed with professionals of diverse fields. Our outreach events at the Center of Science and Industry (COSI), Boonshoft Museum of Discovery and WestFest at the Ohio State University allowed us to educate kids and parents about the goals of our project while promoting the ideas of synthetic biology. Our discussions with professionals guided our approaches to both dry-lab and wet-lab during brainstorming and conceptualization. Other professionals gave us great direction in the proposed implementation of our project and made us more aware of our project’s place in the world, specifically for its potential use as a therapeutic for GI cancers. Additionally, we obtained valuable data on the experiences of GI cancer patients and their perspectives on our project idea.

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#### Wet Lab

Unfortunately, due to struggles with securing a lab space, many of our wet-lab goals had to be delayed. We still made sure to document our expected plans and wet lab engineering process for future reference. The work we did with dry-lab and human practices should prepare our team to pursue a similar project with a heightened focus on wet-lab activities. Better yet, our work could be used by other iGEM teams to develop a project of their own using T3SS.

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