An important part of iGEM is collaborations. For us, the collaborations served to demonstrate whether we could feasibly incorporate microfluidics and our system TERRA into the synthetic biology workflows of other lab groups. In return, we provided these other teams with a more automated method of performing some of their necessary tasks. During this iGEM season, we collaborated with the BostonU, Harvard, and MIT iGEM teams.
Droplet GenerationThis chip was designed to encapsulate sodium alginate, calcium chloride, and cells suspended in media in droplets for later use in the Harvard iGEM team’s keratin skin patches. The Harvard team needed to find a way to control pore sizes of alginate, which were used to suspend E. coli and would only allow the desired treatment to be secreted through the pores. Our microfluidics-based system offered a controlled and precise method that would alter the amount of E. coli in each alginate suspension droplet, as well as change the pore size with of the alginate gel with calcium chloride, due to the features of a microfluidic chip. Our teams conducted literature review to design a microfluidic device to provide an solution for the Harvard team’s project.
Since this chip passed the proof of concept experiments using colored water, this was the chip used, in combination with the TERRA Adapter during our collaborative meeting with Harvard on August 9, 2018. The chip was tested with biology a total of 4 times, each time eventually leaking before creating a stable stream of droplets. An analysis of the failure concluded that pressure around the chip was ineffective at keeping the requisite area around the channels sealed given that there was too much empty space between the channels and the pressure
applying binder clips. The future chip should either be smaller in overall area or the channel design should be spread out over a larger portion of the chip area. Harvard iGEM left us with the material needed to attempt to encapsulate their droplets on our own for when the new chip is assembled and tested.
Following the observations made when testing the initial version of this collaboration chip, the overall chip area was significantly reduced while the actual design was made more spaced out. This, combined with a thicker layer of PDMS, lead to the chip seal holding. The chip was capable of creating the proof of concept droplets out of different colors of water, but when the different solutions were introduced, the viscosity of the sodium alginate solution inhibited the formation of droplets. Now that the chip no longer leaks, it was decided that perhaps shortening the length travelled by the segregated solution would reduce the effects the viscous solution.
This version of the Harvard Collaboration Chip was not designed by the 2018 BostonU Hardware iGEM team, but by one of our mentors Ali Lashkaripour. It is similar to last version with a few differences. First, the three liquids meet right before the droplet generator. Second, the overall dimensions of the chip are smaller which leads to a better seal. Lastly, the channels have varying widths due to the initial purpose of chip. This last detail is not relevant to the chip’s purpose here. When this chip was tested with the sodium alginate solution, it behaved similarly to to the last version, with the viscous solution creating tailing droplets but no individual encased droplets. After observing
this repeatedly under a variety of different flow rates ratios, it was decided that a new geometry was needed to achieve the collaboration’s desire outcome. Using a similar chip with only a single input, we were able to conclude that the while the sodium alginate solution was capable of forming droplets, it did so independently of the chip geometry. After this test and some literature review, a T-junction was decided upon for the new version of this collaboration chip.
This latest version of the chip utilizes a T-junction instead of the droplet generators we have been using in the rest of our chips. Similar to the previous chip, all three inputs meet right before the T-junction. Unlike the other chips, a T-junction uses only a single stream of oil instead of pinching the solution with two streams, letting the droplets formed partially independent of geometry. During the first test with parameters similar to those tested in the last design, the problem remained. While there were some droplets being formed independent of chip geometry, there remains no way of making consistent, uniform droplets with the viscous sodium alginate solution. Additionally this chip, like the other
versions, is prone to clogging to due to the mixing of the calcium chloride and sodium alginate solutions, making this chip inefficient to test.
While the chip did not completely work with the final design, we learned about the effects of viscosity on droplet-based microfluidic systems during the four chip iterations and subsequent weeks of testing. If we were to continue this project, the next step would be to perform a more in-depth study of the effects of viscosity on the generation of droplets in microfluidic devices by experimenting with various flow rates and chip geometries. Click here to find the fabrication instructions and SVG files for this chip:
Northeastern iGEM (NEGEM)
Our team hosted the Northeastern iGEM Conference (NEGEM) with the BostonU iGEM team. On July 6, 2018, the iGEM teams from Harvard, MIT, UCONN, BostonU Wetlab, and BostonU Hardware teams met at Boston University to present their work and share advice among teams. Each team presented within time constraints of the Jamboree, with 5 minutes allocated to set-up, 20 minutes for the presentation, and 5 minutes for questions. We created feedback forms for each team in order to create a simple and organized method of collecting feedback. NEGEM gave our teams a great method of obtaining
Serial DilutionInspired by the InterLab Study, this series of chips attempts to follow the protocol of running three consecutive 1:20 dilutions followed by a pair of consecutive 1:10 dilutions. These sequences of mixers are then paired with an valve actuated output selection mechanism such that either the third, fourth, or fifth dilutions can be selectively dispensed onto an agar plate. Since the real estate on our desktop CNC milled microfluidic chips is limited, this protocol has been divided into two chips which, when run in tandem, accomplish these specific serial dilutions. The first chip in the system consists of three consecutive mixers, design with flow rates intended to handle the three 1:20 dilutions. The other chip will run the two 1:10 dilutions and the output selection mechanism.
This initial chip consists of four inputs, one for the initial sample and the other three are used to dilute that initial sample by adding water. Following each addition of water are mixer so that the liquids are mixed into one homogenous, dilute solution. While the chip works in theory, for this design to work, the initial flow rate of the initial sample is 0.1 or 0.01 mL/hour. This slow rate alongside the larger dimensions of these channels made using this chip unfeasible. And it was redesigned with smaller features.
In an attempt to solve the problem of multiplying flow rates faced by the last chip, this version tried to siphoned off three quarters of each dilution through varying resistance. The hope was to mitigate the dramatic increase in flow rates further downstream by only allowing a portion of the flow to continue through the chip, thus allowing faster initial flow rates then those used in the previous chip design. Unfortunately, by creating a path with an intentionally lower resistance, all the flow was redirected to this path instead of just 75% intended to follow this path. This left no fluid to continue through the rest of the chip.
This chip is similar in design to that of the original version of this chip with the two primary differences being smaller feature sizes and the addition of more outputs and accompanying valves. The point of smaller feature dimensions is to decrease the wait time, even when using the small flow rates used in the initial chip design. The valves are used for two purposes: testing and timing. This chip was tested with a member of the 2018 BostonU iGEM team using a green fluorescent protein as the initial sample in order to determine the accuracy of each dilution. The valves leading to the additional output ports provides
manipulation of the fluid and the ability to collect a sample after each dilution instead of just the third. After testing the system, relying on visual cues to facilitate each dilution, it was determined that the some math modeling needed to be done to determine the timing needed for each mixer to reach a steady state before collecting each of the three outputs. Click here to download the fabrication documentation and SVG files for this chip: