iGEM 2022 — Team bostonu-hw

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{% block title %}Notebook{% endblock %}
{% block lead %}A complete history of the AM1 in the making from its engineers.{% endblock %}
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May

# Motivation, Brainstorming, Research

The month of May allowed the team to meet each other, get aquainted with the space, as well as further investigating the problem we are aiming to solve.

## Week 1: May 23-2022

- Team conducted safety lab training
- Team met for the first time
- Toured our lab space
- Began preliminary research
- Simple design/technology demonstrations
- Familiarization with all the facets of iGEM

## Week 2: May 30-2022

- Continued research
- Began brainstorming system and its properties

* 4 Main Aspects:

+ Liquid Environment
+ Sampling Methods
+ Incubation
+ Sensing

- Very basic functionality and prototyping:

* Pressure/Syringe Pumps
* Sampling System Tubing
* Simple Filtration System

June

# Prototyping, Testing, Demonstration

The majority of June was still part of the creative process, however the team was able to further specify some of our initial designs by including attributes such as materials and dimensions.

## Week 3: June 6-2022

- Designed initial aquatic environment with prototype of filtering system
- Designed second iteration of the sampling system

* Began to incorporate automation via Fluigent pressure pump libraries

- Investigated different heating elements and regulatory components for initial incubator prototype
- Finalized the use of microcontrollers and raspberry pis

* Decided what aspects of the system will be automiated via the pi

- Began to reach out and touch base with other Boston based iGEM teams

## Week 4: June 13-2022

- Idea to improve modularity of the system with more user-design
- Further improved sampling system to reduce the footprint

* Decreased the number of syringe pumps
* Investigated creating a purely pressure based system

- Proposed two different iterations of the incubator setups
- First of 2 collaborations with the MIT iGEM team

* Visited their lab space and discussed our project ideas for the competition as well as other ideas for all the deliverables
* Read more on our [Collaboration](https://2022.igem.wiki/bostonu-hw/collaborations) page

## Week 5: June 20-2022

- Finished the 3D modeling for the proposed aquatic environment
- Compared logistics of the new and old pressure pump system

* Decided on purely pressure based system

- Designed first microfluidic device for droplet generation
- Finalized the heating element for the incubator

* Decided on using a cooling chip in reverse

- Completed training for microfludiic fabrication

## Week 6: June 27-2022

- First of several updates with the PI Dr. Densmore

* Communicated updates and projected goals for the rest of the summer

- Further refined presentation skills for events upcoming in July
- PI's Synthetic Biology Course

* Read about our contributions to this course on the [Education](https://2022.igem.wiki/bostonu-hw/education) page

July

# Education, Connection, Outreach

July was a major opportunity for outreach, allowing us to spread the word of Synthetic Biology to a variety of different communities and audiences.

## Week 7: July 4-2022

- Final filtration prototype developed
- Continued to implement modularity into the system

* Decided to include peristaltic pumps for environment simulation

- Pressure system automation began via Arduino microcontrollers
- Incubation trials concluded, proceeding to final prototype
- SSH into raspberry pi for complete system automaion
- Began promotional video preparations

## Week 8: July 11-2022

- Decided on a logo design
- Began to layout system for final housing production
- Second round of fabrication demonstrations
- Prepared for following education outreach events
'

## Week 9: July 18-2022

- Held two different education outreach sessions

* Discussed Synthetic Biology basics as well as Bioethics
* Read about each of them in the [Education](https://2022.igem.wiki/bostonu-hw/education) section

- Began producing the final housing
- Incubator completed
- Automation begins to be tested via the raspberry pi

## Week 10: July 25-2022

- Housing was finalized

* Finalized materials for frame, flooring, housing

- Storyboard for promotional video compelted
- Fabricated the first microfluidic device

August

# Building, Testing, Refining

As the end of the summer approached, it was time to kick things into high gear. A majority of final building took place as well as major progress on the rest of the deliverables.

## Week 11: August 1-2022

- Housing construction continued
- Began design of the web applictaion for automation

* Easy, user-friendly interface to controls the parameters of simulation
* Web app to be deployed on a touchscreen for ease of use

- Began updating graphical abstracts
- Held an educational outreact program at BioBuilder

* Read more about it in our [Education](https://2022.igem.wiki/bostonu-hw/education) page

- Hosted MIT for the second half of our collaboration

* Read more about it in our [Collaboration](https://2022.igem.wiki/bostonu-hw/collaborations) page

## Week 12: August 11-2022

- Met with Processing Engineer at Deer Island Waterwater Treatment Facility

* Led to modifications in housing via a new multifluid input system
* Read more about it in our [Human Practices](https://2022.igem.wiki/bostonu-hw/human-practices) page

- Continued working with touchscreen and web app compatibility

## Week 13: August 16-2022

- Met with Biosensor Collaborators

* Read more about it in our [Human Practices](https://2022.igem.wiki/bostonu-hw/human-practices) page

- Remote team began working on our wiki

* Distributed and assigned pages to team members
* Made minimal aesthetic decisions
* Compiled all work to a single location

- On site team worked on the final stages of housing and automation

* Decided how to house and connect final electronic components

- Planned commercial content

## Week 14: August 22-2022

- Added minor adjustments to housing
- Remote team continued working on our wiki

* Wrote content for pages
* Began researching and making stylistic choices

- Filmed commercial content

## Week 15: August 29-2022

- Remote team continued working on our wiki
- Submitted commercial video
- Finished final housing

* Added and connected final tubing and wiring

- Fabricated microfluidic chips

September

# Enhancing, Collecting, Finalizing

With the start of the fall season, our team began finalizing our system and its delivarables, including working on our wiki, final presentation, and documenting content.

## Week 16: September 5-2022

- Team met in person again!!
- Fabricated final microfluidic chips
- Began running demos in housing setup

* Generated droplets using our sampling system

- Continued wiki development

## Week 17: September 12-2022

- Began working on final presentation
- Continued running demos and collecting data

* Ran colored droplet demo

- Continued wiki work

## Week 18: September 19-2022

- Continued final presentation edits
- Continued wiki work
- Continued running demos and collecting data

* Ran fluorescence demo

## Week 19: September 26-2022

- Began rehearsing final presentation
- Added final plots and images to wiki

October

# Rehearsing, Presenting, Perfecting

By October all the team had left was final tweaks to the wiki, finishing our presentation and rehearsing for the Grand Jamboree.

## Week 20: October 3-2022

- Final wiki tweaks ahead of freeze
- Practice presentation with live audience

## Week 22: October 10-2022

- All done with wiki!!
- Final presentation practice and rehearsals with live audiences

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{% block lead %}Traverse the team's trials and tribulations through the engineering design process.{% endblock %}
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* Engineering Success
  [Engineering Success](#Success)
  [Filtration System](#Filtration)
  [Multi-Stage Filtration](#Final)
  [Multifluid Input](#Multi)

# Engineering Success:

Throughout the course of our project, there were several design changes that came as a result of the Design – Build – Test – Learn cycle. From fabricating several microfluidic chips with different input/channel sizes to 3D printing multiple iterations of our filtration component, we definitely learned a lot from our multiple design changes. We kept note of each change, because just as we learned from the shortcomings of our earlier designs, future iGEM members might benefit from seeing what worked and what didn’t.

# Filtration System - 3D printed prototypes:

Our liquid filtration system was very important for the later components of the testing process because of the sensitivity of the microfluidic chips. With such small channels, even microfibers could clog the system. We solved this problem by designing a multi-stage filtration system. Our multiple design changes came from needing a watertight system (something our 3D printed parts couldn’t provide) and ease of access to the user (allowing for the removal and replacement of filter paper for continued product usage). We updated our CAD files throughout the process and also 3D printed a few iterations to get a better understanding of how they would fit into our system. Below are some of the designs ranging from the initial brainstorming stages to our final design concept.

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/1sttank.png)

Figure 1.A:

Filtration Tank

Filtration for large debris occurs in the middle of the two tank system

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-successs-figure2.jpg)

Figure 1.B:

Filtration Device

Slanted Design would provide issues when going to machine final design

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure4-new.jpg)

Figure 1.C:

Filtration Device V2

Incisions for seamless tank insertion

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure5-new.jpg)

Figure 1.D

Filtration Device V2.1

Circular Filter for compatibility with easily accessible filter papers

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/updated-pic.jpg)

Figure 1.E

Filtration Device V2.2

Two 3D printed iterations of different thicknesses

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure6-new.jpg)

Figure 1.F

Filtration Device V2.5

Sliding Mechanism to provide access to system from side panel

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure7.jpg)

Figure 1.G:

Filtration Device V3

Sliding mechanism to provide access to system from system side panel

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure8-new.jpg)

Figure 1.H

Filter V3.5

Incisions for sensors that run from top to bottom of tank

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/filtrationsystem.png)

Figure 2: Comparing the different filter designs

# Final Multi-Stage Filtration System:

It was at this point we realized that using one tank for filtration and regulation of liquid parameters was unideal. In design V3, you can see that the sensors need to pass through the filtration piece (due to length of sensors). This in turn created waterproofing issues. Because of our project’s heavy reliance on electronics like microcontrollers and Fluigent pressure pumps, it was essential for us to prevent even the slightest of leaking liquid. We turned to a new system that incorporated 3 tanks to solve this issue. The diagram below shows our final system.

This final design incorporates both a horizontal and vertical filtering process. In the initial filtration system, the centerpiece filters for large debris like rocks and pebbles. The liquid is then sent to the simulation tank where sensors and side reservoirs (attached above the simulation tank) allow for sensing and regulation. Because the reservoir liquids have not been filtered yet, the final mixed solution is passed to the secondary filtration and sampling tanks where small amounts of solution are filtered using a sliding mechanism. It was because of our multiple iterations during the prototyping process that we were able to utilize multiple filters for a more comprehensive filtration system. Below you can find the engineering design process we went through; what started with one design resulted in various iterations all from which we learned a lot.

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/designprocess2.png)

Figure 3: Engineering Design Process

# Multifluid Input Design:

To sense and regulate different liquid parameters like pH and salinity, we needed access to small water reservoirs of various solutions (water with high and low pH for example). The issues occurred when trying to determine a way to get precise solutions out of reservoirs into our regulation tank. We needed to be able to move exact amounts of liquid. Our first design had reservoirs directly above the regulation tank; the idea was to use motorized valves to open and close output from the reservoirs. But from testing the reservoirs themselves, it was immediately clear that excess solution would be mixed into the tank due to gravitational forces. From researching and testing different water pumps, we came across peristaltic pumps that allowed for pumping water at fixed flow rates. We no longer needed gravitational forces to move our water.Our final design incorporates reservoirs attached to the sides of the regulation tank and peristaltic pumps moving the liquids between them.

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure11.jpg)

Figure 4.A:

Multifluid Input V1

Initial multifluid input design. Holes on bottom relied on gravity for dispensing liquid solutions

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/eng-success-figure12.jpg)

Figure 4.B

Multifluid Input V1

Initial multifluid input design. Holes on bottom relied on gravity for dispensing liquid solutions

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/eng-success/2ndtank.png)

Figure 4.C

Multifluid Input V2

Changed design to incormorate peristaltic pumps (controllable flow rates). Simpler design (geometrically easier to assemble)

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{% block lead %}Examine the results and metrics of the AM1's detection capabilities.{% endblock %}
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* Results
  [Results](#Results)
  [Aquatic Environment](#Aquatic Environment)
  [Microfluidic 1](#Microfluidic Device 1)
  [Incubator](#Incubator)
  [Microfluidic 2](#Microfluidic Device 2)
  [Touchscreen](#Touchscreen/User Interface)
  [Housing](#Housing)
  [Automation](#Automation)
  [Replication](#Replication)
  [Future Plans](#Future Plans)

Beyond the data collected for the specific experiments detailed in our [proof of concept section](https://2022.igem.wiki/bostonu-hw/proof-of-concept), the main results include the protocols established and levels of integration in this system.

# Aquatic Environment:

○ The aquatic environment filters water and simulates key characteristics of a chosen aquatic environment. Currently this step includes the simulation of a desired pH and water turbulence, via a rotating magnetic system. The [proof of concept](https://2022.igem.wiki/bostonu-hw/proof-of-concept)  test for the aquatic environment tested the pH simulation, but this feature can be extended to include other sensors such as salinity, ORP, and temperature. These key characteristics are important for testing biosensor efficiency in different environments.

○ In order to operate the peristaltic pumps, sensors, and fan from the raspberry pi, the code to actuate these components was integrated into the python environment.

○ Additionally, the [multi-stage filtration system](https://2022.igem.wiki/bostonu-hw/engineering)  has two distinct filtering steps to remove large debris and smaller fibers that can clog a microfluidic chip.

# Microfluidic Device 1:

![](https://static.igem.wiki/teams/4485/wiki/wiki-items/results/ayastable.png)

Figure 1: Droplet Generation with Variation in Pump Parameters

○ The first step for operating the droplet generation microfluidic device is sampling from the aquatic environment and priming the fluid through the tubing to reach the microfluidic device. This table describes the pressures, flow rates, and time required to accurately generate droplets.

○ The microfluidic device can consistently mix the biosensor and water sample and create droplets of the same size, as shown in our [proof of concept page.](https://2022.igem.wiki/bostonu-hw/proof-of-concept)

# Incubator:

○ The incubator was modularly constructed with a heat block that holds 1.5 mL and 2.0 mL test tubes. Due to the modular nature of the incubator, the current heat block can be replaced with one that holds 0.5 mL or 15 mL test tubes depending on the output volume from the droplet generation microfluidic device.

○ A closed-loop protocol controls the power supplied to the thermoelectric chip based on the temperature sensed inside the incubator. This code is actuated by an arduino that is controlled by a raspberry pi. This infrastructure combining the analog abilities of the arduino and the computing power of the raspberry pi is a key feature of the system.

○ Lastly, the incubator heats up quickly (< 30 minutes) and [maintains the temperature during the entire incubation period.](https://2022.igem.wiki/bostonu-hw/proof-of-concept)

# Microfluidic Device 2:

○ A key goal of the second microfluidic device is to reinject the droplets and control their speed and rate. This is achieved by spacing out the droplets with oil to ensure the droplets move through the sensing region and a rate the sensor can sample.

○ The second goal for this microfluidic chip is to integrate to the CIDAR Lab’s established fluorescence sensing equipment. We show that this [flourescence sensing](https://2022.igem.wiki/bostonu-hw/proof-of-concept) can be incorporated into the system.

# Touchscreen/User Interface:

○ A touchscreen interface was incorporated into the system for users to easily interact with the device. Specifically, the touchscreen has sliders to set variable levels for the device and protocol saving features to enhance the user experience.

○ The touchscreen connects to the raspberry pi which controls and actuates various protocols at the touch of a button.

# Housing:

○ The modular housing setup can hold the different components in a variety of layouts to optimize the space for specific experiments.

○ Because the system is composed of both liquid and electronic components, we divided the product into two halves - one solely for liquid inputs and the other for electronics and microfluidics.

# Automation:

○ The AM1’s automation is incorporated into each stage of the device. A raspberry pi controls all the overall scheduling of the individual protocols.

○ The user’s settings are used in an automated protocol that controls the aquatic environment. The user sets specific parameters and the closed loop simulates the desired environment by controlling the sensors submerged in the water and the peristaltic pumps that dispense liquid until the target liquid parameter levels are reached.

○ For the first microfluidic device, the sampling, priming, and droplet generation have all been characterized and occur automatically at that stage of the automated protocol.

○ The incubator is controlled by a closed-loop arduino code that sets the power level of the heating chip based on the sensed temperature.

○ Lastly, automated fluorescence sensing has also been incorporated into the protocol as a proof of concept for sensing the output from a biosensor.

# Consideration for Replication of Project:

○ The timings and pressures in the microfluidic automation protocol are specific to the microfluidic devices, tubing lengths, test tubes, and biosensor used in these demos. To use the automation protocol without any adjustments, the exact setup described in the proof of concept and results pages will be needed.

○ The fluorescence output of the droplets with IPTG-inducible GFP bacteria was measured with CIDAR Lab’s existing fluorescence sensing setup.

# Future Plans:

○ At this stage of the aquatic environment, the pH and turbulence features have been tested, but we’re interested in expanding this to other sensors including salinity, ORP, and temperature.
Currently, certain protocols within the automation have been tested thoroughly, but further testing using different components is necessary for robust automation.

○ Additionally, we want to test other biosensor protocols. Once such protocol might require two separate incubation steps allowing for single cell encapsulation in droplets, growing up these cells through droplet incubation, piconinjecting the aquatic sample, incubating again, and reinjecting into the fluorescence chip.

○ Finally, we would like to incorporate different biosensors with other types of outputs. To accomplish this goal, the integration of fast-sampling electronic sensors in the sensing microfluidic device is required.

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