How To Make A MEGA-plate

This page provides step-by-step instructions for the MEGA-Plate experiment for evolving and tracking microbial evolution on antibiotic gradients (Baym, et al., Science, 2016b)

This guide is divided into sections:

Design of a typical MEGA-plate experiment

• Example design of regions on the plate (Here for example: 6-step Chloramphenicol gradient experiment).

Assembly of a MEGA-plate

Materials (for a 62x82cm plate)

• Polycarbonate for the base of the plate:
  • Plexiglas sheet 30cm X 60cm X 0.3cm
• Polycarbonate for the walls of the plate
  • Polycarbonate sheet 62cm X 82cm X 0.6cm
• Heated glass for the lid
  • Alternatively acrylic sheet for the lid (superior optical clarity to polycarbonate)
• Sci-Grip adhesive (if not available, Dichloromethane works -- you just need an appropriate solvent for welding acrylics)
• Syringe and Needle- For spreading the glue directly on the right area.

Setting up a MEGA-plate experiment

Materials

• 9 two-liter jars or Erlenmeyer flasks (appropriate for autoclaving)
• Agar (http://www.bd.com/ds/productCenter/214010.asp#)
• LB Broth (http://www.bd.com/ds/productCenter/240220.asp)
• Appropriate pipettes for adding antibiotics to media
• Blowtorch (e.g. a creme brûlée torch)
• Useful, but not strictly necessary
  • IR touchless thermometer

Procedure

• Sanitize the plate by filling it with a dilute bleach solution (~5-10%) overnight
• Dump out bleach solution, do not wipe out residual traces.
  • Optional: further sanitize with UV light for around 1 hour. (This is not clearly necessary, and some plastics can leach out reactive compounds when exposed to UV.)
• Prepare 9 liters of agar, either in 2L jars or Erlenmeyer flasks:
  • 5 of them with 2L with 2% LB+Agar.
  • 2 of them with 2L with 2% Agar.
  • 2 of them with 2L with 0.27% Agar.
• Do autoclave 40 min in 121c twice.
• When the bottles are cooling put the plate in the designate place.
• After the liquids are cold enough add the antibiotics.
• Pour the first layer to the right well and let it solidify.

• Pour the second layer all over the plate and let it solidify.

• Pour the third layer all over the plate and let it solidify.

• Inoculate the bacteria.

Procedure for building the plate

• Use a table saw to cut the frame and the bottom of the plate from the XXX.

• Use laser cutter to cut the gradient barriers for your design:

XXX

• • 8 pieces of 40cm X 1.27cm
  • 4 pieces of 40cm X 2cm
  • 2 pieces of 60cm X 2cm

• Glue the By using syringe and needle glue the pieces like the plan design:

Draw the design of the plate on it.

Put the cut parts in the right place.

Glue them by syringe and needle containing dichloromethane.

• Check for leaking and fix if needed by re-gluing.

Procedure for imaging the plate

• Should you wish to take time-lapse movies, we have had success imaging at regular 10-15 minute intervals for the duration of the experiment

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.

References

Relevant papers and books

1. Baym, M., Lieberman, T. D., Kelsic, E. D., Chait, R., Gross, R., Yelin, I., & Kishony, R. (2016). Spatiotemporal microbial evolution on antibiotic landscapes. Science, 353(6304), 1147-1151. doi:10.1126/science.aag0822[1]

Contact

This page was developed by Michael Baym and Rotem Gross from the Kishony Lab.

For questions, please contact:

1. Michael Baym (mhbaym@gmail.com)
2. Rotem Gross (rotemgross1@gmail.com)
3. Roy Kishony (rkishony@technion.ac.il)