This week we were tasked with designing a milli-fluidic device that could be used for our project. I'm really excited by the idea of a lab on chip(s) so I came up with three small chips that could accomplish some of the tasks i'll need to take on for the final project. The different chips include an assembly chip which could be used for Gibson Assembly, a media chip to test varying medias and fructose concentration, and a color chip to test the light sensitive system and evaluate visible and UV color ranges.
Milli-Assembly
Milli-Media
1-Find a research or journal article where researchers cultivate 2 of more microbial strains. What technology are they utilizing? How scalable is this approach to more than 2 strains? How do they address issues related to requiring multiple media?
I looked into the gut-on-a-chip, where they used a microfluidic device to cultivate eight different probiotic strains in direct contact with living human villi for periods of a week or more. They also successfully co-cultured non-pathogenic E.coli with villi. Previous models only allowed co-culturing for short periods of time, typically less than a day. The main problem seemed to be that the microbial cells overgrow and compromise intestinal barrier function. The chip overcomes this by recreating physiological conditions such as peristalsis through mechanics and fluid flows. Through flow, they were also able to flow a constant supply of nutrients to both bacterial and villus epithelial cells while removing unbound gut bacteria increasing viability.
The system does seem scalable as long as some of the challenges are taken into account including: determining optimized incubation times which vary across microbial cells according to their ability to adhere, the seeding density which can cause outgrowth if excessive, and flow rates which also depend on microbial species.
While ordering plasmids for the final project I noticed that they light sensing system is consistent across. I am using the YF1/FixJ photoreceptor. However I keep changing the reporter to different variations of amilCP or amilGFP. So the first chip would explore the possibility of doing Gibson assembly on a chip! You would insert the inputs including the backbone and Gibson assembly mix that would be divided amongst all samples. The reporter sequences would then be inserted and the mix in a snaking channel. The mix would then flow to an incubation area where the chip could be incubated at adequate temperatures and then flushed out to evaluate the plasmid assembly. Based on a device by Patrick, William G., et al.
Another area that needs study is the various media types and concentration. This chip is based on a gradient distribution. In one inlet you insert the bacteria, another for media, and a final port for a sugar. Based on the bacteria distribution you can then evaluate the favorable conditions since the bacteria will tend to grow towards that direction. This is useful to get a range, without filling up a 96 well plate. Based on a chip by Atencia, Javier, Jayne Morrow, and Laurie E. Locascio.
PART A: MILLI-FLUIDIC DEVICE
PART B: MILLI-FLUIDIC DEVICE
MILLI-ASSEMBLY
MILLI-MEDIA
Microfluidic Device Design:
Patrick, William G., et al. "DNA assembly in 3D printed fluidics." PloS one 10.12 (2015): e0143636.
Atencia, Javier, Jayne Morrow, and Laurie E. Locascio. "The microfluidic palette: a diffusive gradient generator with spatio-temporal control." Lab on a Chip 9.18 (2009): 2707-2714.
Ching, Terry, et al. "Fabrication of integrated microfluidic devices by direct ink writing (DIW) 3D printing." Sensors and Actuators B: Chemical 297 (2019): 126609.
Microbiome:
Kim, Hyun Jung, et al. "Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow." Lab on a Chip 12.12 (2012): 2165-2174.
Kim, Hyun Jung, et al. "Co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device." JoVE (Journal of Visualized Experiments) 114 (2016): e54344.
Goers, Lisa, Paul Freemont, and Karen M. Polizzi. "Co-culture systems and technologies: taking synthetic biology to the next level." Journal of The Royal Society Interface 11.96 (2014): 20140065.
Shah, Pranjul, et al. "A microfluidics-based in vitro model of the gastrointestinal human–microbe interface." Nature communications 7.1 (2016): 1-15.
2-One of the great challenges in microbiology is culturing "unculturable" microbes. Propose a methodology for how you might explore this significant space of uncultured microbes.
In class we talked about the difficulty of culturing anaerobic microbes. I also discovered that there are different levels of oxygen tolerance. Some microbes prefer anaerobic conditions but are not affected by the presence of oxygen, while others find oxygen to be toxic. With challenges of building large chambers or containers I was wondering if you could culture anaerobes and perhaps also study the effects of gradients of oxygen at the same time.
Surely I found a chip that did just that. The HuMIX model (human-microbial crosstalk) was able to co-culture human epithelial cells (Caco-2) with an anaerobe (LGG). They maintained anoxic conditions by continuously bubbling the medium with dinitrogen gas. Only when they inoculated the plate did a small about of oxygen seep in. Could this system be replicated for other anaerobic experiments where anaerobic conditions are maintained chemically in a microfluidic device?
3-Review an article on an artificial gut-on-a-chip technology. What scientific hypothesis are they testing with this in vitro tool? Could you propose an upgrade or innovation to their technique to enable the exploration of other scientific hypothesis? Provide an example of at least one hypothesis you would explore with your proposed system.
I reviewed an article on the gut-on-a-chip mechanically active microfluidic device. They were testing whether they could use microfluidics to develop a physiologically relevant in-vitro model of the human intestine which could undergo peristalsis, experience fluid flow, and support the growth of microbial flora without compromising human cell viability. Through mechanics to recreate peristalsis and fluid flow they were able grow columnar epithelium that grew into folds and form a high integrity barrier that mimics whole intestine systems.
One interesting area to explore is co-culturing with anaerobes. The gut microbiome contains an incredible array of different bacterial strains and some are anaerobic. Therefore testing is needed to understand the gut microbiome in anaerobic conditions. In other words, what are the effects of anaerobic microbial interactions in the gut microbiome? There are researchers working in this field including Shah, Pranjul, et al. and the HuMix model. To do so they used three chambers - a perfusion microchamber, a human epithelial cell culture microchamber, and a microbial culture chamber. They maintained anaerobic conditions by continuously bubbling the medium with dinitrogen gas.
I'm curious if taking on a similar approach and adding a modular component to the gut-on-a-chip technology Kim, Hyun Jung, et al. can be studied to further refine the system beyond a two layer chip.This can be used to test the impact of anaerobes in in the gut and overall whole body health if the chips are connected.
Gut-on-a-Chip, Kim, Hyun Jung, et al
Gut-on-a-Chip, Kim, Hyun Jung, et al
The HuMIX Model, Shah, Pranjul, et al.
REFERENCES
Milli-Color
The last chip could be used to evaluate expression of the various chromoproteins. Blue light could be focused on the central chamber to test the Yf1/ FixJ system. Multiple inlets and outlets would also make it possible to simultaneously test various reporters.
While reading more on color I also became aware that some insects such as bees can see UV light. Flowers, such as the one shown on the top right might appear yellow to us, but to the insect a landing pad is drawn! I am curious about how the various reporters also show in visible light vs UV and am thinking of incorporating a symbiotic element so that not only humans benefits from the proposal. Based on a chip by Ching, Terry, et al.