iGEM 2022 — Team ohiostate

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April

## Wet Lab

#### 04/16/22

- Brainstormed ways to build upon the Lipid A Phighter project from last year
- Decided upon a diagnostic approach using phage
- Built off of a conversation with Dr. Ahmer, who identified bacteriophage as a rapid way to indentify bacteria

## Dry Lab

#### N/A

## HP

#### 04/06/22

- Brainstormed HP ideas and looked at past winners of human practice awards
- Began searching for potential sepsis contacts

May

## Wet Lab

#### 05/14/22

- Focused on setting up a permanent iGEM space for future teams
- Secured funding from CFAES and ordered a set full set of microbio laboratory equipment
- Secured permanent location in professor Rowan's lab, but couldn't be set up because of supply chain delays and issues

#### 05/ 28/22

- Began researching strong promoter sequences: a strong promoter would translate more quickly, creating more fluorophore, thus allowing sepsis to be more quickly diagnosed

## Dry Lab

#### 05/18/22

- Selected four projects to pursue
- Phage-Bacteria population model
- Bioreactor design
- Cellular automata model to show the benefits of a phage cocktail
- Sepsis detection device as a physical model that can detect fluorescence and report it

#### 05/25/22

- Each project was further planned out and it was decided what would be in and out of scope for each

## HP

#### 05/17/22

- Discussed summer camp ideas to make it more specific to our project and sepsis
- Looked into potential collaborations with local teams to have a summer camp or event at science museums

#### 05/24/22

- Developed Instagram posts for general synthetic biology education

June

## Wet Lab

#### 06/24/22

- Found a lab space in Dr. Wood's lab in the chemical engineering department
- Transported equipment from the lab used last year (microbiology department) to the new lab
- Set a timeline for PCR: Satvik perform PCR over the weekend and then do cleanup and train Elijah next week
- PCR clean up next week

#### 06/27/22

- Satvik finished PCR cleanup with Elijah and gel extracted all protein inserts for use in phage cloning

## Dry Lab

#### 06/08/22

- Split into teams to tackle each project concurrently
- Preliminary research was conducted by each team for their selected project

#### 06/15/22

- Each team presented preliminary research for their respective projects
- Presentations were analyzed and next steps for each project discussed

#### 06/22/22

- Assigned research roles for each project
- Began looking into different professionals that could help provide insight into our bioreactor design

#### 06/29/22

- Researched optimal bioreactor design based on optimal pH, temperature, and agitation conditions
- Discussed the ideal type of bioreactor, either a stirred tank or airlift

## HP

#### 06/10/22

- Corresponded with Wright State iGEM team to plan events at COSI/Boonshoft

#### 06/13/22

- Ryan, Emily, and Angela met with the University of Washington iGEM team to discuss our 2021 project and share both 2022 projects

#### 06/23/22

- Ryan, Fesume, and Angela video called with Wright State to form a collaboration and create more specific plans for COSI/Boonshoft

July

## Wet Lab

#### 07/06/22

- Elijah completed nanodrops
- Satvik determined nucleic acid profiles recorded were not sufficient to continue with cloning
- Satvik switched wet lab's focus to this year's clones
- Promoter research led Satvik to use constitutive promoters which would not be inhibited by the bacteria

#### 07/16/22

- Satvik, Elijah, and Ankit are still the only members allowed to work in Dr. Wood's lab
- Satvik ordered parts from IDT so he could begin on this year's construct

#### 07/31/22

- Satvik performed a miniprep on the lab-provided backbone but failed
- Have to re-streak and attempt to re-isolate the backbone

## Dry Lab

#### 07/06/22

- Brainstormed questions to ask bioreactor specialists
- Sent emails out to these specialists

#### 07/13/22

- Discussed current bioreactor design
- Began research regarding each bioreactor's medium and how pH and temperature could be controlled
- Began thinking about wiki design and coding

#### 07/20/22

- Discussed resarch regarding medium and controls and how to apply it to the bioreactor
- Basic SolidWorks bioreactor model was created and displayed

## HP

#### 07/13/22

- Angela met with Christina Harsch to understand the Ohio Department of Education standards
- Christina also helped with creating our lesson plans for our outreach events

#### 07/21/22

- Ryan, Emily, and Angela spoke with William Lynes about his challenging sepsis experience
- Dr. Lynes described the lack of sepsis knowledge among patients and doctors in the hospital
- From this meeting, started to brianstorm some ideas to combat this lack of knowledge

#### 07/28/21

- Ryan and Angela discussed an outreach project with Laurie Miller from COSI and the Wright State iGEM team
- Decided on October 9th for the event at COSI and finalized what synbio experiments we would do
- Strawberry DNA lab, Phage puzzle, fluorescing bacteria, and a microscope activity were designed

#### 07/31/21

- Angela talked with Christina Harsch about cognitive benefits of 2D versus 3D puzzles to help us design the phage puzzle
- Decided that a 2D pahge puzzle would engage more types of learners
- Ryan worked on the [protocol](https://static.igem.wiki/teams/4341/wiki/education/phage-bacteria-matching-game-protocol.pdf) for the phage puzzle

August

## Wet Lab

#### 08/06/22

- Satvik is having trouble isolating the backbone and is now the only current team member in the lab
- The backbone was determined to be contaminated after 2 weeks of isolation attempts

#### 08/16/22

- Satvik isolated the backbone and begane ligation process
- Ligation and transformation process carried out until we have viable colonies with our contruct cloned in

#### 08/29/22

- Backbone was used up after only 2 ligation attempts
- Satvik had to reisolate to ensure the backbone was fresh and not contaminated (took about 3 days to get back on track)

## Dry Lab

#### 08/03/22

- Fesume presented his finished population model
- Discussed potentially using the model to predict the population of phage that is cultivated in a bioreactor

#### 08/10/22

- Emily presented further research into how to quantify fluorescence intensity using the sepsis detection device
- Could use image sensors to take a picture of the petri dish and then use software to calculate intensity
- Could use Beer's law and look at absorbance of fluorescence

## HP

#### 08/02/22

- Angela created a sepsis awareness poster to put in hospitals to teach patients about sepsis and how to prevent it
![](https://static.igem.wiki/teams/4341/wiki/hp-page/what-is-sepsis.svg)

#### 08/15/22

- Angela updated activity protocols for the science museums

#### 08/21/21

- Angela reserached ICU pyschosis and post sepsis syndrome

#### 08/26/21

- Fesume, Angela, Ryan, Elijah, and Kaya recorded the promotional video

September

## Wet Lab

#### 09/10/22

- Satvik is still attempting ligations for bacterial clones
- Attempt 3 and 4 failed: after consulting with Sophia and Nate, Satvik re-streaked and re-isolated the backbone to ensure he was using fresh DNA for his ligations
- The grad students recommended that Satvik do this before all future attempts
- Attempts 5 and 6 also failed: Satvik used a newer T4 ligase buffer from New England Biolabs in case an expired buffer was to blame
- Thus far, all attempts proved negative, with every attempt having no colony growth

## Dry Lab

#### N/A

## HP

#### 09/10/22

- Fesume 3D printed the 2D phage puzzle to use at Boonshoft and COSI
![](https://static.igem.wiki/teams/4341/wiki/notebook/bacteriophagepuzzle.svg)

#### 09/25/22

- Angela, Fesume, and Ryan traveled to the Boonshoft Museum alongside Wright State to teach listeners about DNA, synbio, and sepsis
![](https://static.igem.wiki/teams/4341/wiki/education/puzzle-osu.svg)

October

## Wet Lab

#### 10/02/22

- Satvik continued to attempt cloning with no success
- Attempt 7: grad students suggested using a backbone isolated from a different strain
- Swtiched from pET28a backbone to a pET29a backbone

#### 10/09/22

- Attempt 7 failed, leaving wet lab without results or proof of concept

## Dry Lab

#### N/A

## HP

#### 10/09/22

- Angela and Fesume traveled to COSI with Wright State to educate visitors about synthetic biology and sepsis

#### 09/25/22

- The strawberry DNA protocol was posted on Instagram to allow others to conduct this experiment at home

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#### **Bacteria-Phage Population Model**

The OhioState 2021 team created a steady state population model and that can be found
[here.](https://2021.igem.org/Team%3AOhioState/Model)
This year, we attempted to remove the steady state assumption from our population model. To do that we contacted Dr. Lenski and he suggested the Runge-Kutta method to solve the system of ODEs.

Two iterations were completed with the population model, one where the concentration of free phage (P’) and uninfected bacteria (S’) were set equal to the concentration of free phage (P) and uninfected bacteria (S) at time t. However, this iteration produced results that were incorrect, so another iteration was run to fix the issue. For the next iteration, P’ and S’ were set equal to P and S at t-τ, where τ is the latent period of time. More information about the population model can be found
[here.](https://2022.igem.wiki/ohiostate/model)

#### **Cellular Automata Model**

In talking with Dr. Strathdee, author of *The Perfect Predator* and Co-Director at the Center for Innovative Phage Applications and Therapeutics, we saw how important phage cocktails are. If one only administers one type of phage or phage that all target the same bacterial receptor, there is a high risk the bacteria mutate or evolve to be resistant to that phage. However, using many different phages can keep selective pressure on the bacteria and help stop it from mutating away from the phage. We created a python model to visually explain this complex topic, by using cellular automata to show how a bacterial colony develops in the presence of either one or two types of phages. More information about the population model can be found
[here.](https://2022.igem.wiki/ohiostate/model)

This model was not created as is right away. Instead it was created over many iterations. The first draft of this model tested one phage type and one bacteria type. Once this model was shown to run successfully, a second type of bacteria and phage were coded in. With two phage and two bacteria in the model we could finally represent a basic phage cocktail. Each bacteria will be assumed to be different, whether one type evolved from the other or if they are simply different. The two phages will act upon each bacteria of the same type only. When this model was constructed and ran, Figure 1 resulted.

![Bad phage model](https://static.igem.wiki/teams/4341/wiki/engineering/actual-bad-model-cropped.svg)

Figure 1. First Code Iteration Results with Non-Overlapping Phage Populations

A weird pattern in the data is seen. If the two phage heat maps are overlapped, they essentially fill out the whole grid. So no type 1 phage are getting into the areas where type 2 phage are. Looking back at our initial assumptions provides a clue. One says that only one type of phage can surround a bacteria at a time. This is not realistic, phages can surround any type of bacteria, their types only impact what bacteria they can **infect**. In order to fix this, the phages were allowed to occupy the same space. That is, both phage type 1 and type 2 could be surrounding the same type of bacteria; however, only the phage that could infect the bacteria actually went on to infect. In this way, the phages could spread throughout the grid space, infecting and killing as much bacteria as they could. Figure 2 shows the updated results and the lack of cohesiveness between the two phage maps.

![Good phage model](https://static.igem.wiki/teams/4341/wiki/engineering/cocktail-model-good-cropped.svg)

Figure 2. Second Code Iteration Results Featuring Overlapping Phage Populations

#### **Wet Lab**

As we were starting our protocols for cloning our phage DNA, we did not want to spend valuable time correcting easily avoidable mistakes made in previous iterations of our project. We contacted Dr. Brian Ahmer to talk about our plans for our constructs. Based on our talk, we resolved to use both the T5 and pR promoters to highly upregulate protein production of our construction. Here, we took the lac operon out of the T5 promoter to remove a limiter in its operation. In addition, we also made a rrnB-pR fusion promoter after our own research to find something that *E. coli* cannot suppress.

At first, we planned to use luciferase, however after the OhioState 2021 team had a few troubles regarding its expression, we decided to use a much stronger fluorophore in mCherry. Using this, we hoped to have the strongest possible expression in which to compare our promoters.

One of the decisions that must be made when deciding to make the phage reporter is promoter design. So we decided to experimentally test 4 reporters and test them in another reporter assay for the promoters.

We wanted to have the strongest possible promoter sequence for our phage reporter, so our diagnostic time would be as fast as possible. The team researched some common strong promoters, and specifically liked the idea of a constitutive promoter, which contains fewer or no inhibiting sequences. We then designed 4 different parts. We ordered the required sequences to spec, and began the cloning process.

![Wet Lab Engineering Flow](https://static.igem.wiki/teams/4341/wiki/engineering/wetlabengineeringflowcherry.svg)

Figure 3. Promoter Design Process

First, we isolated the backbone from pET28a to give a basis so we could test our diagnostic construct. Then, we began ligating the sequences into the backbone, and hit our first issues. After 2 attempted and failed ligations, we didn’t have enough backbone to continue.

We consulted the graduate students in Dr. Wood’s lab, as they are very experienced with ligations and know ways to improve on further iterations. They recommended that we reisolate the backbone for all following attempted ligations. However, even once we implemented this change there was still trouble: the transformations weren’t taking. No colonies containing our specific construct had survived the transformation process. Despite this, we reconfirmed the size of our four inserts, resulting in the gel below showing that they were the approximate sizes that were expected.

![gel result](https://static.igem.wiki/teams/4341/wiki/engineering/gel.svg)

Figure 4. Gel Electrophoresis Result: Lane 1 rrnB P1; Lane 2 pR; Lane 3 pR-rrnB P1; Lane 4 T5-rrnB P1

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#### Find our wet lab experiments in the below pdf! They can also be downloaded [here.](https://static.igem.wiki/teams/4341/wiki/experiments.pdf)

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### Wet Lab

Initially, the wet lab wanted to test the OhioState 2021 team's constructs from last year in phage for phage therapy (Phighter Phage) and test a new set of promoters to find the best turnaround times for a phage reporter (Phinder Phage). However, because of not finding lab space until late June, we decided to focus solely on the testing and design of the Phinder Phage. Furthermore, restrictions on phage cloning until August were put in place because of worries about contamination with an existing project in the lab space. This meant we decided to pivot and focus on just testing the best promoter for our Phinder Phage. We were successfully able to isolate pET28 backbone via gel extraction and isolate our four promoter constructs fused to mCherry as seen in the gel below.

![gel result](https://static.igem.wiki/teams/4341/wiki/engineering/gel.svg)

Figure 1. Gel Electrophoresis Result: Lane 1 rrnB P1; Lane 2 pR; Lane 3 pR-rrnB P1; Lane 4 T5-rrnB P1

However, when we went to ligate and transform, no colonies were growing on our plates. We decided to swap out ligases but this proved ineffective. Then, we decided to re-streak the origin for our backbone and attempt ligation with that new backbone. Again, this proved ineffective. Further attempts were made by changing buffers and re-making plates. In the end, they also proved unsuccessful and because of time constraints, work in the wet lab had to stop. While investigating the cause of these ligation failures after our final attempt, other graduate students who just started ligations told us they also noticed issues with plating ligation transformation. This leads us to believe the problem may be because of temperature control issues on our incubator and dry heat baths.

In the future we hope to actually clone our reporter fusion into *E. coli* and then pick the strongest one for further work and study. Then, we would clone this reporter construct into various phages and test the time it takes for detection to occur.

**Successes:**

* Isolation of backbone (pET28a)
* PCR and digestion of four fusion with mCherry
* Selection of hybrid promoters to test from literature
* Ligations of backbone and insert

**Failures:**

* Not able to clone into bacteria
* Not able to clone into phage

### Human Practices

The human practices committee was able to educate and create materials to inform people about sepsis and synthetic biology. Talking with doctors and other professionals helped the team highlight which important effects and causes of sepsis were largely unknown by the population. These highlighted areas led the team to research ICU psychosis from sepsis patients, post sepsis syndrome, and other detrimental impacts of sepsis. Understanding some of the most important effects of sepsis allowed the team to inform listeners. Our visitation of two Ohio science museums gave us an opportunity to enlighten others to learn about synthetic biology. The museums also allowed us to teach listeners specifically about our project and why it is so important to combat sepsis.

Although our team had great successes with outreach, we had hoped to speak with a greater number of doctors who deal with sepsis on a daily basis. It is important to learn how doctors are adjusting to new technologies as well as how they are combating increasing sepsis cases. A lack of responses from professionals restricted our ability to analyze sepsis stories. Also, because of struggles with acquiring a lab space and other lab difficulties, we were unable to update our Instagram followers on the progress of our experiment. Despite not being able to skillfully use Instagram to teach about sepsis from our lab updates, we were still able to post an at home synthetic biological protocol as well as sepsis facts.

**Successes:**

* Created sepsis awareness fliers to place in hospitals
* Traveled to COSI and Boonshoft science museums to teach listeners about sepsis and synthetic biology
* Spoke with doctors and patients who helped us narrow down the most important effects of sepsis
* Posted sepsis facts as well as the Strawberry DNA experiment protocol to allow viewers to repeat the experiment at home

**Failures:**

* A small sample of doctors were spoken to because of a lack of responses
* The team had hoped to highlight progress from lab work on Instagram, but difficulties from acquiring a lab space and other complications shifted the content of the posts

### Dry Lab

Dry lab completed three models for our project: the phage-bacteria population model, the cellular automata, and the bioreactor model. The population model was able to build on OhioState 2021’s population model. The previous year’s model assumed that the interactions occurred at steady state, which would not be applicable to all situations. This year, the model was created using the Runge-Kutta method and was able to account for unsteady state interactions. The cellular automata displayed that phage cocktails were more effective than using a single phage at eliminating bacteria. For the bioreactor model, we were able to create a dynamic model for the concentrations of phage and *E. coli* leaving the bioreactor.

In the future, we would like to expand on our current models. Currently, the bioreactor model only includes a dynamic model for concentration; however, there are a number of other parameters that will need to be modeled to create a control system for the bioreactor. For example, temperature would need to be modeled to ensure the contents within the bioreactors are maintained at the temperature that leads to the most phage growth. Using the dynamic models, the team would be able to make the control systems by converting the dynamic models into transfer functions. With the control system created, our goal would be to build the bioreactor and test how well our control system works. The population model would need to be tested versus experimental trials to understand how accurate it is. With the experimental data, we could learn ways to improve the population model. Similarly, we would want to run experiments to display that our findings with the cellular automata are valid and work to eliminate as many assumptions as possible.

**Successes:**

* Cellular Automata displayed that phage cocktails were more effective at eliminating bacteria compared to just using a single phage
* An unsteady state phage-bacteria population model was successfully made utilizing the Runge-Kutta method
* A dynamic model for phage and *E. coli* concentration leaving the bioreactor was created
* Successfully designed and printed the 2D phage puzzles
* Expanded the phage database to include 4 more sepsis-causing-bacteria

**Failures:**

* Our team attempted to create a sepsis detection device. The device would analyze a blood sample and determine how much the blood fluoresces when phage is added. We were planning on quantifying how much the sample fluoresces to determine how much bacteria is in the sample. However, we did not have enough time to create a presentable model for the device.
* Initially, our goal was to build the bioreactor. However, our team lacked the resources or manpower to do so. Also, many of the researchers and professionals we reached out to were unresponsive which limited our progress at points during the summer. Therefore, towards the end of the summer we scrapped that idea and focused on creating a dynamic model of the concentration leaving the bioreactor.

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