Lab Implementation
To validate our project in the laboratory, we planned several experiments to evaluate our design choices and investigate the functionality of the genetic construct.
How much supplemental iron can the plants tolerate?
To determine iron tolerance, we would grow wild-type plants at increasing concentrations of FeSO4, to find the highest dosage of iron at which viability is not significantly decreased. This will help us evaluate if we can supplement the plant media with iron for our engineered root magnetotropism.
Does our clinostat actually cause the expected misdirected root growth phenotype?
To understand if growing wild-type plants in our clinostat changes their root phenotype, we first need to observe the roots of wild-type plants in natural gravity to record what normal growth is and then compare it to growth in the clinostat. Additionally, we discussed with Dr. Collin Timm that we could grow a mutant with a known disordered root growth phenotype as a positive control for our plants grown in our clinostat. In response to Dr. Timm’s recommendations, we searched for starch-free mutants. We chose an ADG1 mutant [1] and a PGM mutant [2]. ADG1 and PGM are key starch granule synthesis genes [3]. Without the accumulation of starch, the statoliths will not sediment under the force of gravity. In these mutants, the role of statolith in sensing gravity should be eliminated causing a disruption of the root gravitropism and a disordered root growth phenotype under natural gravity.
Does our engineered root magnetotropism work to recover proper directed root growth?
After we transform the wild-type plants to overexpress ferritin, we would grow the engineered plants on iron supplemented media plates in the clinostat with the magnetic field generator engaged. Also, we could transform the starch-free mutants with our engineered plasmid to determine whether ferritin loading could have any effect on the disordered phenotype of the mutants in gravity.
Which transit peptides cause localization of ferritin to statoliths?
We would use fluorescence microscopy imaging to check for ferritin localization to the statolith.
Real-World Implementation
In an actual spaceflight system, a pair of Helmholtz coils would surround the root laterally. The root would be placed and grown in an appropriate substrate suitable for water diffusion and air circulation, and efficient nutrient delivery. The Helmholtz coils would surround this substrate. Drs. Simon Gilroy and Richard Barker suggested consideration of recyclable mediums such as clay or a suitable plant substrate to substitute for the arselite used in current systems (see Human Practices). The engineered (with ferritin) plant roots would respond to the gradient magnetic field and grow in the direction of the field (downwards).
Material diffusion models were hence developed to study the transport of several crucial nutrients and molecules for plant growth through plant growth mediums. Our preliminary theoretical lumped-model results suggest that transport is less efficient in recyclable mediums (which is corroborated by research). This suggests that if MS buffer is used with a recyclable medium in space (ie. extending our lab setup to a different medium that is more practical for space agriculture applications), nutrients will be transported relatively well, though not as efficiently as they will be in agarose. Therefore, we can see that (i) different mediums could work and (ii) optimizing surface area of plant roots could be of interest in recyclable mediums.
The constant current coil driver and plant growth incubator would likely occupy an Express Rack aboard the ISS. The major concerns for adaptation of our existing electrical system to such an Express Rack, according to McCollum et al.’s 2020 paper “Electromagnetic Compatibility Considerations for International Space Station Payload Developers,” [4] are:
- Electromagnetic Interference (EMI)
- Power Sources
- Data Integrity
When operating in switching mode, our device can produce electromagnetic interference at the switching frequency (0-500 kHz), but the resulting electrical fields are easily attenuated with an aluminum enclosure. Magnetic fields are of greater concern, and we may need testing to determine what thickness of Mu-metal is appropriate for magnetic shielding in addition to the aluminum enclosure.
As for power, our current implementation utilizes an AC/DC converter and true earth chassis ground. AC power on the ISS is provided via an inverter, and subsequent rectification of this AC power introduces significant inefficiencies. The ISS has 28V DC power available and we can instead use a more efficient 28V DC to 5 VDC converter to supply power to the system. Our existing wiring schematic isolates the 5V supply from the chassis, and we would have to do the same on the ISS.
Our current device uses an I2C bus for communication. I2C is designed mostly for intra-board communication and is susceptible to noise, and is less suitable for inter-board communications like between the RP2040 controller and constant current driver board peripherals. For applications like these we have included microcontroller alternatives into the constant current driver board design - we would instead use the QtPy RP2040 as opposed to the ATTiny412. This would allow us to take advantage of the QtPy’s USB for noise-resistant communications over noise-shielded cables and comply with ISS standards without making any modifications.
End Users
End application of our project is targeted towards engineering plant seeds prior to spaceflight missions using our designed construct such that the plants cultivated in spaceflights under microgravity conditions can be responsive to magnetic fields. Studying plant growth, development and metabolism is the prime focus area of the NASA Plant Biology program, and therefore, we envision our experimental setup to be tested through the program at the International Space Station (ISS) - designed to test the growth of a variety of new plants for spaceflight crew. To validate our prototype in a real-world environment, its effect would also have to be tested on Earth under gravity and under simulated microgravity ground controls at the Kennedy Space Center, as also done by the Plant Biology program.
To ensure the health of our astronauts, the next and most essential step before spaceflight implementation would be to examine the nutritional composition of plants grown in space, as well as look at the microbiome of plants in orbit. Once validated, the prototype will benefit long spaceflight missions by enabling growth of edible produce in space that could be used as a sustainable source of fresh food by crew on the ISS, as well as for long-duration space-flights.
Lastly, as mentioned in the Project Description, local farms and businesses having interest in vertical farming would also benefit from our prototype. Maryland itself is home to a significant agricultural community that includes 12,400 farming operations [5]. Several local farms and businesses have a niche in vertical farming, including Bowery Farming Nottingham and City-Hydro. We further explored this implementation through our Human Practices outreach, in particular our discussion with Dr. Paul B. Thompson. Dr. Thompson is a Professor of Agricultural Ethics at Michigan State University. Throughout his career investigating agricultural ethics and the role of scientific technologies and recombinant DNA in agricultural practices, Dr. Thompson has met with several farmers and other stakeholders in agriculture. He pointed out that on-Earth implementations for space projects often have a lot of promise because they can be translated directly into solutions. Further, Dr. Thompson remarked that the agricultural sector is always aiming to optimize productivity and efficient use of agricultural land, so potential implementations of our project to optimize and speed up plant growth would certainly be of commercial interest if proven to be effective. This can be attributed to the “technology treadmill” phenomena in agriculture, where stakeholders are constantly implementing new technologies to improve productivity and stay ahead of their competition.
Safety and Other Considerations
One of our project’s products is a genetically engineered Arabidopsis thaliana / Brassica rapa capable of responding and orienting root growth in the direction of external magnetic field gradients. Arabidopsis thaliana, a small flowering plant belonging to the Brassicaceae family and is adopted as a generic model organism considering important features such as its short generation time, ability to produce many small seeds, self-pollination capability and its small genome size which simplifies genetic analysis and manipulation [6]. However, according to the National Park Service, an agency of the US Department of the Interior, A. thaliana is listed as an invasive species in North America. A. thaliana is not indigenous to the US, and is only native to Asia, Europe and North Africa. Considering its potential to rapidly displace native plants and/or significantly alter natural ecosystem structure and function, if it were to escape the lab, it could contribute to decrease in plant biodiversity. To mitigate this concern of accidental dispersion outside the lab, we worked closely with experts at the Johns Hopkins Applied Physics Lab to learn and follow appropriate plant growth conditions, as well as instigated lab access controls in place.
Brassica rapa is a rapidly-cycling crop plant, commonly used in teaching laboratories. Protocols for agrobacterium-mediated transformation of B. rapa are readily available and well optimized. Moreover, B. rapa was also selected owing to its self-incompatible characteristics. This mechanism serves as a barrier for intraspecies pollination for the plant, by preventing self-fertilization in the same plant or in case of B. rapa, other plants of the same species [7]. In our case, it served as an important line of safety since self-incompatibility of a genetically engineered B. rapa would serve to prevent pollination between our engineered B. rapa and other Brassicaceae plants present in the actual spaceflight plant cultivation system.
In order to generate strong enough gradient magnetic fields to pull down engineered statoliths, we had to build our own constant current coil driver. The major risk this device poses is an electrical shock hazard. The first and most effective safety measure we have taken is to fully enclose the device in an enclosure with the chassis grounded to true earth. In the case of any fault or short, the current would be safely discharged. We have also implemented fuses at (1) the IEC outlet on the device enclosure and (2) the constant current coil driver board. These fuses would prevent damage to the circuitry should the current consumption of the device exceed a safe limit for each of the 2 driver boards or the system as a whole, and would also trip following a short or other fault. All of our wiring and connectors are rated for at least 1.5 times excess the maximum currents of our device. Finally, the fully enclosed nature of our device prevents accidental contact with any of the components.
Challenges in Real-World Application
There are some other considerations that would have to be taken into account. Firstly, the produce produced using our engineered plant would possibly be considered a GMO. This would create legislative concerns and possibly restrictions for genetically-modified plants to be cultivated aboard the ISS. However, on the positive side, the VEGGIE project by NASA has been successful in cultivating genetically-modified lettuce capable of producing human parathyroid hormone, to help prevent bone loss in spaceflight crew [8]. This sheds light on the acceptance of GMOs aboard the ISS and hopefully acceptance of GM crops by spaceflight crew and astronauts. Spaceflight crew would essentially have to carry transgenic seeds aboard the ISS and cultivate the plant there, creating concerns revolving around the safety and efficiency of the transgenic seeds and plants. The steadily increasing acceptance of GM food by people globally, particularly in the European Union, would support a positive change of the legal framework surrounding testing and cultivation of GM crops [9], opening opportunities to successfully implement our engineered plants in the real-world.
Another potential challenge we would have to encounter if our project were to be translated to a real-world application would be generating safety data through animal and human testing, and clinical trials to prove that our GM plant is safe for human consumption. During clinical trials, we would also have to look into nutritional aspects along with addressing safety considerations.
References
[1] https://abrc.osu.edu/stocks/148810
[2] https://abrc.osu.edu/stocks/414
[3] Zhang, Y., He, P., Ma, X., Yang, Z., Pang, C., Yu, J., ... & Xiao, G. (2019). Auxin‐mediated statolith production for root gravitropism. New Phytologist, 224(2), 761-774.
[4] M. McCollum, L. Kim and C. Lowe, "Electromagnetic Compatibility Considerations for International Space Station Payload Developers," 2020 IEEE Aerospace Conference, 2020, pp. 1-9, doi: 10.1109/AERO47225.2020.9172800.
[5] USDA/NASS 2021 State Agriculture Overview for Maryland
[6] Woodward, A. W., & Bartel, B. (2018). Biology in bloom: a primer on the Arabidopsis thaliana model system. Genetics, 208(4), 1337-1349.
[7] Kitashiba, H., & Nasrallah, J. B. (2014). Self-incompatibility in Brassicaceae crops: lessons for interspecific incompatibility. Breeding science, 64(1), 23-37.
[8] Genetically Modified Lettuce May One Day Help Space Travelers Fight Bone Loss | Smart News| Smithsonian Magazine
[9] Ichim, M. C. (2021). The more favorable attitude of the citizens toward GMOs supports a new regulatory framework in the European Union. GM Crops & Food, 12(1), 18-24.