Team:BostonU HW/Collaborations

BostonU_HW

Collaboration
Collaborations

Harvard

Cell Culturing Microfluidic | July - September 2017 | Harvard iGEM

This collaboration between the BostonU Hardware Team and the Harvard iGEM team was based around engineering a microfluidic system to culture cell and measure the resulting cell growth. Each team was responsible for separate parts of the system. The BostonU Hardware Team primarily worked on the design, manufacturing, and testing of the microfluidic device. The Harvard iGEM team primarily worked on an optical density sensor which was aimed to measure cell growth. Even though each team focused on individual parts of the system collaboration was imperative to test and develop each teams work.

Stage 1

The initial stage of the collaboration focused on outlining the goals we wanted to achieve from the collaboration. This meeting occurred over the summer after initial interest was sparked after hearing about each other's work at a local conference. The Harvard iGEM team had initially wanted to design the chip in order to do culture fiber producing cells. After looking over the initial designs that the Harvard team produced, both teams agreed that more literature review was needed to redesign a new microfluidic chip.

Stage 2

After reviewing different cell culturing microfluidics devices, we had come across a microfluidic chemostat and turbidostat which matched the same functionality that both teams were looking to achieve. The teams agreed that this design was able to be manufactured and suitable for performing the cell culturing experiment.

The two main challenges that were involved with replicating the microfluidic chip were designing the microfluidic device and manufacturing the PDMS membrane with the proper thickness. An initial chip was designed by the BostonU Hardware team using circular chambers. After performing several tests, it was determined that the paper’s original design would be more suitable. The next step was to obtain the proper PDMS. The PDMS membrane needed to be extremely thin (75um thickness) in order to be actuated the full depth of the chamber. This prompted the BostonU Hardware team to learn how to spin coat PDMS in order to obtain the precise thickness. There was varied success in the beginning, but eventually we were able to get a consistent result. However, after subsequent testing we realized that more research was still needed.

We then reached out to one of the publishers of the paper, Rajeev Ram, who is a professor at MIT University. We wanted to have an up close look at the device after realizing the high level of complexity it took to design and manufacture the chip. We went to visit his lab and see a copy of the microfluidic device as well as the corresponding system that is used to run it. Both teams were provided with valuable insight into the chip’s operation and design after the visit. This allowed us to understand the minimum pieces of the design that needed to be kept in order to maintain the same functionality.

Stage 3

The BostonU Hardware team worked to develop and manufacture a replication of the microfluidic device. Eventually with the help from the Harvard iGEM team we were able to replicate a relatively accurate design of the chips cell culturing chamber. However, when the chip was tested using colored water there was very little success. The fluid was unable to move in a proper clockwise movement and was not able to be displaced fully from any chamber. Progress was eventually stopped due to the lack of hardware resources from last year's project, Neptune. In order to continue the development of the microfluidic module more work would be needed with programmable syringe pumps.

Stage 4

The last portion of the collaboration was to validate the Harvard iGEM team’s optical density sensor. The BostonU Hardware team developed small scale microfluidic chips in order to test the sensor. However, in order to test the OD sensor, the Harvard iGEM team vapor polished the surface to remove any etchings that was left from the manufacturing process. This aimed to decrease the possibility of inaccuracies that could occur during testing. The Harvard iGEM team came to the BostonU Hardware team’s lab. Check out the Harvard iGEM team's wiki for more information.

Harvard

Fluorescence Microfluidic | July - September 2017 | Boston University Wet Lab iGEM
This collaboration was based around co-engineering a microfluidic chip that performs the BostonU Wetlab team’s Toe-hold protocol. This chip was then integrated into the MARS Chip Repository as the fluorescence chip. Our preliminary fluid functionality checklist was used while testing the final chip design in order to receive feedback regarding its effectiveness. Feedback was also provided for the Microfluidics 101 content and the usage protocols for our microfluidic chips which was used to improve MARS.

Stage 1

The initial stage of the collaboration focused on determining what goals we would aim to achieve over the course of the summer. A key component of this meeting was a discussion regarding transferring the BostonU team’s biological protocol onto a chip. If possible, we wanted to automate their entire testing procedure on a single chip and have five samples prepared for plate reading simultaneously. The second objective of the collaboration was to have this design incorporated into the MARS Repository and be accessible for iterations or adaptation even after the collaboration ended. With this in mind, we moved on to the design and test stage.

Stage 2

Before beginning the design stage, we wanted to ensure that our microfluidic chips could be read within a plate reader. Following a consultation with the synthetic biologists working in the BDC, there were no obvious detriments to using polycarbonate inside a plate reader. In order to test this, we milled a mini chip with the same well spacing and depths as a 94 well plate. This was inserted into a 3D printed skirt and placed inside the plate reader. GFP was filled inside the milled wells of the chips in various patterns and the plate reader’s results analyzed for any discrepancies. The results indicated that the chip could be read accurately toward the center of the chip with increasing inaccuracies at the periphery. This was incorporated in the future designs.

The initial designs for this chips focused on a few key features:
  1. Using shared inputs to decrease the chip’s complexity
  2. Utilizing valves in order to create a complete seal between chambers
  3. Determining how to measure volume of liquid dispensed
  4. Avoiding leaks to prevent contamination

After a number of iterations, another meeting was held with the BostonU Wetlab team in order to discuss progress on the design. It was concluded that performing all five tests on a single chip would not be feasible given the time constraints. As a result, their biological protocol was adapted to suit the limits of our chip design abilities. This included cutting down the samples run to a single test.

The final design integrated a chamber designed to fit the dimensions and depth of a well from a 96-plate reader. It also utilized our new metering primitive in order to ensure accurate ratio volumes of liquid would be dispensed while running the protocol. In order to make the design easier to use for the BostonU team, the control layer’s inputs were shared as much as possible.

Stage 3

The final stage of the collaboration was having the BostonU team test the final iteration of their chip design. Prior to visiting our lab, they were sent our Microfluidics 101 educational material, assembly protocols and chip usage protocols to review. This was in order to simulate the experience of a synthetic biologist using MARS to understand how our chips work.

Following this, their members practiced assembling and testing two existing microfluidic chips - PCR and DNA Digestion. Over the course of this they were able to feed back on the relevance of the educational material we had provided them with. This feedback was extremely valuable as it came from synthetic biologists with limited understanding of our hardware. Their feedback was noted and then integrated into our existing workflow. Finally, the BostonU Wetlab team assembled, tested and graded their chip design using a preliminary Fluid Functionality checklist. The results they gained through this testing, and the ability of the checklist to accurately represent the failures during the test, have been used to improve our grading system.

The chip design has since been added to the MARS Repository under “Fluorescence” and has been categorized as Quantification. For more information regarding the chip’s design and documentation, refer to its chip page on our Wiki.

Lead Assay Microfluidic | July - September 2017 | WPI iGEM

Our collaboration with WPI involved designing a microfluidic device that could perform a portion of their lead assay. This chip would use primitives designed by the BostonU Hardware team such as metering. Working on this chip emphasized the importance of coengineering microfluidics and synbio.

Stage 1

We first met the WPI iGEM team at NEGEM1, an event hosted by BostonU that invites New England iGEM teams to present their project plans and ideas to other teams. We discussed the potential for a collaboration between our two teams, and soon after we contacted each other via email. During our first Skype call we discussed some potential ideas for their collaboration. We decided that the goal of the collaboration would be to design a chip that could perform their lead assay. With this in mind, they sent us a PDF copy of their full protocol and we began planning a visit to BostonU. For more details regarding this assay, please visit the WPI iGEM team Wiki.

Stage 2

WPI’s visit to BostonU had two purposes. First, it would help validate WPI’s assay protocol and results in another lab, Second, it would give our team a better understanding of the protocol we would be moving onto a microfluidic device. Two students from WPI, Catherine and Aylin, met with a member of our team in Boston University lab on July 26th. Before performing the experiment in the lab, the WPI team gave a quick overview of the experiment as well as any specific safety protocols required while working with lead. In the lab, the WPI team members performed their protocol in its entirety; the BostonU Team member observed but did not touch any biological materials.
During this process, the two teams discussed various limitations and difficulties found in performing the assay by hand. A difficulty of note was the importance of adding and mixing reagents at exact times. For example, GSH needed to be added between 15-20 seconds after mixing the Lead and Gold Nanoparticles; however, adding the GSH at a uniform time was extremely difficult. With this information in mind, we decided that reliable timing would be an integral part of the microfluidic chip design.
WPI performing their lead assay in the BostonU lab on July 26th 2017. They performed their entire assay in the lab while the BostonU HW team observed and asked questions.


Stage 3

After meeting with WPI in the lab, our team had a solid plan for what the design of the chip would look like. The chip would perform one portion of their assay with volumes scaled down by a factor of 10. It would consist of 5 main sections:
  1. Lead Metering: 4.15 ul
  2. AuNp Metering: 3.46 ul
  3. Lead and AuNp Timed Mixing: 17.5 Seconds
  4. GSH Metering: 1.0 ul
  5. Final Mixing: 18 seconds

With this workflow in mind, the chip on the right was designed. The animation details how liquid would flow through the device.

We provided WPI with full documentation of the chip including SVG files, a usage protocol, and the animation seen to the right. All of these files can be downloaded below.




A short animation of how the chip BostonU HW designed would perform WPI's lead assay. Each liquid is colored-coded and the animation is annotated. For more details regarding this chip's flow layer, please refer to the WPI documentation download.