25th of October - David Kong (MIT) and Sean Kearney (MIT)
Presentations and slides
- 9:00 Class starts
'Some of My Best Friends Are Germs,' by Michael Pollan, NYTimes:
'Microbiota-Targeted Therapies: An Ecological Perspective' by Katherine P. Lemon, Gary C. Armitage, David A. Relman, and Michael A. Fischbach
'Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow' by Hyun Jung Kim, Dongeun Huh, Geraldine Hamilton and Donald E. Ingber
'Human Microbiome Engineering: The Future and Beyond'
'How Bacteria Rule Over Your Body – The Microbiome' https://youtu.be/VzPD009qTN4
'Nutritional Psychiatry: Your Brain on Food' https://www.health.harvard.edu/blog/nutritional-psychiatry-your-brain-on-food-201511168626
(1) Culture bacteria from a fermented food (yogurt, cheese, kombucha, kraut, kimchi, etc.). Prepare LB agar plates, and introduce a moistened sterile swab into your fermented food and gently brush the swab on the agar. Incubate at room temperature for 2-4 days or overnight at 37 C.
(2) Co-culture experiments. After colonies have grown, take a picture of the colonies on the plate. Obtain growth curves of in LB media in polystyrene tubes for two morphologically distinct bacterial colonies (use sterile toothpicks to pick colonies into media) grown on the LB agar. Compare this growth curve to that of the same bacterial colonies grown together in LB media. Determine whether the bacteria grown in co-culture have a different growth rate or growth yield than either of the colonies grown in isolation. Note: antibiotic-producing colonies will often have a halo around the colony delineating them from nearby colonies – combinations of these organisms may be interesting to test for their growth facilitation/inhibition effects.
Extra credit: You can identify the bacteria you cultured from your skin by using 16S Sanger sequencing and mapping against a reference database: Genewiz offers a 16S Sanger Sequencing service from bacterial colonies: https://www.genewiz.com/en/Public/Services/Molecular-Genetics/16S-rRNA-Sequencing An extensive protocol can be found here: https://morrislab.wordpress.com/protocols/basic-pcr-including-16s-and-sanger-sequencing-submission/
(3) 3D print a 14 mL culture tube in at least one material. Culture a bacterial strain of your choice (potentially from 1 or 2 or with E. coli as a positive control) in this tube and compare the growth rate (optical density) over time versus a polystyrene control tube. Ideally use a strain featuring antibiotic resistance and culture in the presence of an antibiotic.
Tube and cap design files: http://metafluidics.com/devices/14-ml-culture-tube/
Extra credit: Culture multiple combinations of tube materials and strains, comparing growth rates for each against polystyrene.
(3a) Design a milli- or micro-fluidic 'artificial gut' or other 'organ-on-a-chip' device to be utilized, at a minimum, for cell culture. Feel free to design your device in 2D-CAD software or vector drawing tool (e.g. Adobe Illustrator, AutoCAD) or 3D design tool (e.g. Rhino, SolidWorks).
Rhino (for Mac): https://www.rhino3d.com/
(3b) Fabricate your device, or at least one component of your device. Document the following aspects of fabrication and function in your class page:
What features of your organ are you attempting to emulate? How is your device intended to function? Were you able to fabricate your device? Which components? Which parts 'worked' and which ones didn't? What will you aim to improve for your next iteration of design + build?
Please include photos / screen shots of your digital designs, fabrication process, and final structures!
An example protocol for fabricating an 'organ-on-a-chip': http://www.seas.upenn.edu/~biolines/publications/NatProtoc-13-Huh-protocol.pdf
(3c) Culture the organism from (1) in your milli- or micro-fluidic device. Run a negative control in a device with liquid media only. Collect the liquid culture from your device (+/- bacteria) and plate in the presence of an antibiotic. Report colonies for the +/- experiments.
(4) Share your device designs on 'Metafluidics' (www.metafluidics.org), including Bill of Materials, assembly instructions, and any associated hardware. Irrespective of how far you get in (2), please share your latest iteration! You can always update your device later.
At least one bacterial strain of your choice (ideally resistant to an antibiotic). Liquid media suitable for growing that strain (such as LB). Antibiotic 14 mL polystyrene culture tubes Tool for measuring optical density (spectrophotometer) Petri dishes for plating Bacto Agar (for preparing LB Agar -- add 1.5 g/L of agar to LB broth)
If you do not have access to a spectrophotometer, you can use 'MacFarland Turbidity standards' to indirectly measure optical density.
See: http://biobuilder.org/protocol-a/ Click 'Day 4'.
Also: http://biobuilder.org/wp-content/uploads/2013/11/MiE_00012.pdf Starting bottom of page 267.
3D printer of any kind or use of a service (e.g. Shapeways) Tubing to interface with your fluidic devices Syringes to interface with tubing for fluid handling
Cell Culture in 3D-printed Tube Example
An overnight culture of Escherichia coli was inoculated with a single colony in 5 mL of sterile LB Broth + 50 mg/mL kanamycin and shaken at 37°C. Prior to use, each printed tube was UV-irradiated for 15 minutes. After irradiation, 5mL of sterile LB Broth + 50 mg/mL kanamycin was inoculated with 100 μL of the overnight culture and considered time 0. Tubes were incubated in a 37°C shaking incubator between OD600 measurements.
Optical Density Measurements
An Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany) was used with 8.5 mm centre height uVettes (Eppendorf, Cat. no.: 952010069) with a 100 μL sample volume. Measurements were made at 0, 2, 4, 6, 8 and 24 hours. Samples were diluted 1:5 in media at 6, 8, and 24 hours. All samples were blanked against media incubated in a tube of the given material.