The overall goal of this protocol, is to define standardized approaches for the construction of simplified model plant ecosystems, called EcoFABs, that enable controlled, replicated laboratory experiments to investigate plant microbe interactions that can be further tested in the field. This method can be used to study plant microbe interactions within controlled environments, that allows us to gain detailed mechanistic insights into the functions of micro biomes to allow us to harness them for sustainable agriculture. We helped design this first Eco-FAB, that employs a variety of available 3D printing technologies and to sterilize the plant growth chambers that enable both non-distracting high resolution images and chemical analysis.
The Eco-FAB device developed here is well-suited for the analysis of plant microbe interactions because it incorporates analysis of root morphology and mass spectroscopic imaging. Following the construction of Eco-FAB molds, using 3D printing according to the text protocol, in a disposable one liter container, mix 40 milliliters of siloxane elastomer base with different volumes of curing agent, depending on the experiment. Place the container in a vacuum chamber for at least 30 minutes to remove air bubbles from the elastomer mixture.
Then, remove the mixture, and pour it into the assembled 3D printed plastic mold. In order to generate a uniform PDMS layer, air bubbles have to be carefully removed. Use compressed air to blow off any air residues after transferring the mixture into the mold.
Keep the mold on a heating block at 85 degrees Celsius for four hours. Allow the mold to cool down for five minutes. Then, gently pull out the insert from the mold, and use a utility knife to slowly separate the PDMS from the casting frame.
Press the mold base with PDMS up out of the casting frame. Then, use a knife or other tools to gently separate the PDMS layer from the mold base at the edges, and slowly peel it off from the mold surface. Using a 15 gauge, blunt needle, create an inlet channel on the PDMS layers by making 1.6 millimeter holes in that part.
Then, use scissors to trim the edges of the PDMS layers. To permanently bond the PDMS layers to the microscope slides, use methanol to rinse the bonding side of the PDMS layer at a 7.6 by five centimeter microscope slide, and then blow them dry with compressed air or an ultra-pure nitrogen gun. Place the microscope slide and PDMS layer into a plasma cleaner with their bonding sides facing up.
Close the chamber and the gas vent valve. Then, turn on the vacuum, and pump down the chamber for one minute. Turn on the plasma generator power, and switch the radio frequency, or RF level, to high for one minute.
Then, turn off both the vacuum pump and the plasma power, and vent the chamber to atmosphere. Take out the microscope slide and PDMS layer from the plasma chamber, and quickly press all four edges of the PDMS layer onto the slide with even pressure. Ensure that the center oval region, which is the root chamber, does not touch the slide.
Using ultra-pure water, rinse the Eco-FAB devices. Place one Eco-FAB device in an Eco-FAB container, and add 70%ethanol until the device is submerged. Then, close the container lid and gently shake it to wet all the surfaces inside with ethanol, making sure the root growth chamber of the Eco-FAB device is filled with ethanol, with very few or no air bubbles.
After incubating the devices at room temperature for 30 minutes, pour off the 70%ethanol and replace it with 100%ethanol. Then, incubate the devices for five minutes. Following the incubation, drain off the ethanol, and leave the sterilized Eco-FAB in a Laminar Flow Hood for 16 hours to dry it completely.
If available, sterilize the system by turning on the UV light in the hood for one hour. To carry out seed sterilization and germination, soak the seeds in 70%ethanol for two minutes. Then, with a pipet, remove the ethanol, and use sterile water to rinse the seeds three times.
Next, soak the seeds in 10%bleach solution for five minutes. Then, remove the bleach solution, and use sterile water to thoroughly wash the seeds three times. Following the washes, add sterile water to the seeds, and incubate the tube at four degrees Celsius for seven days.
Evenly spread the seeds on 0.5 Murashige and Skoog, or MS medium, with 0.6%vitagel. And with micropore tape, seal the plates. Then, grow the plants to a root length of about five milliliters.
To transfer the seedlings into Eco-FABs, using a sterile syringe or micropipet and sterile water, flush the root chamber of an Eco-FAB device three times, and then fill the root chamber with the growth medium of interest, such as 0.5 MS medium. Now, carefully insert a single seedling into the plant reservoir of the Eco-FAB device. It is important that the seedlings are transferred carefully from plate to Eco-FAB so that the roots don't break.
Add three milliliters of sterile water into the container, avoiding the Eco-FAB device. This will increase the humidity and reduce the evaporation of the medium from the root chamber. Then, close the container and use micropore tape to seal the lid.
Place the Eco-FAB into a plant incubator, or utilize the Eco-FAB illumination system in a controlled temperature environment suitable for the growth of the respective plant. Then, set the chamber to 24 degrees Celsius. Finally, carry out metabolite profiling and mass spectrometry, according to the text protocol.
These figures show seven-day old Aaabidopsis thaliana, brachypodium distachyon, and panicum virgatum growing in Eco-FABs. Both the dicot and the monocot were found to live up to their reproduction stages in the Eco-FAB. These seedlings represent a group of 14-day old brachypodium distachyon growing in hydroponic medium, as well as sand and soil supplemented with hydroponic medium and water, respectively.
The thin, solid substrate layer and root growth chambers allow light to penetrate through for microscopic imaging of root systems. Here, a brachypodium distachyon seedling was transferred into the Eco-FAB device, and its root structure was recorded by a camera inside a biorad gel imager. The quantification of total root area over the course of three weeks showed a gradual increase at the early stage, followed by a linear growth trend to the end.
In this figure, an Eco-FAB containing the plant growth promoting rhizobacteria, pseudomonas simiae, WCS417 was added into the plant root systems at 10 to the sixth cells per plant. WCS147 microbes colonized the surface of the entire root systems with microbes concentrated around the root tip areas. When refreshing grows media and collecting root exudates from Eco-FABs, it's important to remember to process all the work in a sterile hood to avoid any contamination.
Eco-FAB devices can also be fabricated using a reversible, physical seal by either using a 3D printed clamp or by altering the viscosity of the PDMS. This enables the use of solid growth media and of direct mass spectroscopic imaging. After Eco-FAB development, this system will allow researchers around the world to use these laboratory model ecosystems to study plant microbe interactions using systems biology and imaging technologies.
After watching this video, you should have a good understanding of how to fabricate an Eco-FAB system, as well as its broad applications in ecosystem studies. We hope that this Eco-FAB design will allow other scientists to reach their specific scientific goals. More importantly we hope that other scientists will build on this Eco-FAB design and make it better, and share with other scientists, so that as a community, we can build these laboratory ecosystems to allow us to better understand plant microbe interactions.
Don't forget that working with microbes and sharps can be extremely hazardous, and that precautions should always be taken while performing this procedure.
