Abstract LEGO bricks are commercially available interlocking pieces of plastic that are conventionally used as toys. We describe their use to build engineered environments for cm-scale biological systems, in particular plant roots. Specifically, we take advantage of the unique modularity of these building blocks to create inexpensive, transparent, reconfigurable, and highly scalable environments for plant growth in which structural obstacles and chemical gradients can be precisely engineered to mimic soil.

Citation: Lind KR, Sizmur T, Benomar S, Miller A, Cademartiri L (2014) LEGO® Bricks as Building Blocks for Centimeter-Scale Biological Environments: The Case of Plants. PLoS ONE 9(6): e100867. https://doi.org/10.1371/journal.pone.0100867 Editor: Matthias Rillig, Freie Universität Berlin, Germany Received: April 4, 2014; Accepted: May 31, 2014; Published: June 25, 2014 Copyright: © 2014 Lind et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The work was funded by Iowa State University through a startup grant to LC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Microfluidics[1], and other engineered environments[5], [6] can produce highly controlled micrometer-scale environments for the study of organismal model systems (e.g., mammalian cells). However, scientists or engineers interested in manipulating the environment of cm-scale organisms (e.g., plants) have remarkably few convenient tools at their disposal[7], [8]. This paucity is partly due to the demanding design requirements associated with larger scales (e.g., cost). This liability is particularly evident in the study of plants and their root systems. The development of plants in soil is an important subject of investigation. The provision of food to the global human population is under severe pressure (our supply of food is predicted to be far below demand by 2050[9]) and depends on plant roots[10] (97.6% of global calorie consumption is derived from plants[11]). Roots influence a plant's yield and whether a plant will survive stresses. We know that root growth is strongly affected by its environment, soil, but our mechanistic understanding of these effects is imperfect[10], [12] and strongly limited by technical challenges. Root development is a difficult process to study experimentally. (i) Plants display highly variable root systems, even when genetically identical[13]. (ii) Roots are remarkably sensitive to a variety of stimuli (e.g., gravity, light, touch, moisture, nutrients, oxygen, temperature, trauma, electric fields[14]). (iii) Any volume of soil is unique and impossible to replicate exactly[15], [16]. (iv) Its heterogeneity makes it opaque to most forms of radiation[17]. (v) Its structural and chemical characteristics (i.e., porosity, surface chemistry, nutrient gradients, oxygen gradients, bulk composition, soil biota) cannot be independently manipulated. One approach to avoid this complexity is to characterize the growth of plants in soil-less media, e.g., hydrogels, paper, glass beads, sand. These systems are less inhomogeneous and irreproducible than soil and can be modified – usually to a limited extent – to mimic soil properties such as chemical composition [18], physical structure [19], [20], water availability [21], refractive index [22], or mechanical strength [23]. However, the lack of modularity, versatility, structural precision, and the very limited control over structural and chemical heterogeneities in these systems severely limits the type, complexity, and reproducibility of the experiments they can perform. Microfluidic approaches offer fascinating capabilities for the study of plant roots, but are subjected to limitations in their throughput and in the size of the plants they can host [4], [24], [25]. We here demonstrate that LEGO bricks are highly convenient and versatile building blocks for building cm-scale engineered environments for plant roots. Their modularity enables the fabrication of environments with highly controlled structural and chemical heterogeneities that are suitable for convenient quantitative studies of environmental effects on plant phenotypes[26].

System Design A convenient experimental platform for the study of root development in controlled environments must satisfy a demanding set of design constraints. LEGO bricks, while conceived and sold as toys, satisfy these constraints. Modularity Modular systems can produce many structurally distinct environments from a few different components. Features can be added or removed without remanufacturing the entire experimental setup. LEGO structures are modular. The smallest bricks are 8x8x6 mm. The largest are 48x8x50 mm. The number of different structures that can be made with these units is staggering: six identical bricks can form almost a billion different structures[27]. Scalability Confinement can affect the physiology of an organism[28]. The ability to create experimental platforms of a range of sizes enables researchers to study any plant and their ensembles. LEGO structures can be easily scaled to accommodate different plant species: the smallest enclosed environment that can be produced with LEGO bricks measures 0.35 cm3 in volume, and it is theoretically possible to create LEGO structures capable of containing the largest plant species. Structurally precise Roots are sensitive to the physical structure of their environment. For example, the study of root thigmotropism (the response of a root to touch) requires structures that are of an exact size and shape. The molds used to produce LEGO bricks are accurate to within 5 µm[29], which is comparable to the diameter of a root hair and to the resolution of 3D printing (minimum layer thickness is ∼50 µm in some of the best current models). Capable of increasing levels of complexity A good model system allows for the controlled introduction of experimental variables. LEGO bricks can be used –as shown below – for the generation of physical barriers, air pockets, chemical gradients, and interconnecting chambers to control the growth environment of a plant. Simplicity Simple setups reduce the risk of operator-induced systematic errors. Differently from microfluidic approaches, the assembly of structures from LEGO bricks does not require technical training so undergraduate students can perform LEGO brick-based plant experiments from their first day in the laboratory. Simple experiments that demonstrate fundamental principles of plant growth (e.g., tropisms) or encourage experimental creativity can be conducted by school children of all ages during science education classes[30]. Reproducibility Plant root experimental platforms (e.g. sand columns, rhizotrons, split-root pots) are typically made from scratch. Their reproducibility between labs or across continents cannot be guaranteed. The unique selling point of LEGO bricks is that bricks bought in separate batches are essentially identical and backward- and forward-compatible with each other. Experiments created from LEGO bricks can be accurately replicated anywhere in the world. Affordability The more expensive each experiment is, the fewer experiments can be conducted with finite resources. This fact is especially meaningful in developing nations[31] and in research fields, like plant science, where throughput is an essential parameter. Individual LEGO bricks cost between $0.10 and $1.00 and are sold worldwide. A LEGO structure capable of growing a plant costs $3.1 and is reusable: some LEGO bricks in our lab have been in near-constant use for two years. High throughput The ability to run a large number of experiments at the same time is essential for the establishment, for example, of genotype-environment-phenotype relationships[32]. A LEGO structure like the one shown in Figure 1 can be assembled in less than a minute. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Scheme of the process of carrying out a plant growth experiment using LEGO bricks as building blocks. The same process can be used to prototype and fabricate other biological experiments. https://doi.org/10.1371/journal.pone.0100867.g001 Transparency Twenty eight different LEGO bricks are made from transparent polycarbonate which can be assembled into transparent structures for the real-time monitoring of plant roots over time. Autoclavable Tissue cultures require sterile conditions. Transparent LEGO bricks (with the exception of large base plates) are autoclavable due to their polycarbonate composition: they still fit together in the same way as they do prior to autoclaving and are still transparent after more than 50 autoclave cycles. Opaque LEGO bricks are made from acrylonitrile-butadiene-styrene block copolymer (ABS), and can be sterilized with ethanol or bleach. Three-dimensionality While 2D platforms offer significant advantages in terms of visualization and practicality[33], 3D mediums are more representative of the natural environment of roots[34]. LEGO bricks allow for the creation of nearly arbitrary 3D structures. Chemical inertness Legislative standards ensure the safety to children of LEGO bricks sold in the USA and EU. These standards include maximum soluble levels of toxic or hazardous substances. Compatibility with existing growth environments Tools that integrate with existing experimental platforms are often the most useful. The modularity of LEGO structures enables them to integrate with laboratory protocols e.g., LEGO structures can hold gel, beads, sand, soil, 3D-printed elements, or be structurally precise elements in other setups[35].

Conclusions In summary we demonstrated that LEGO-based environments can (i) scale to the size of the organism under consideration, (ii) allow for real time monitoring of root systems in 3D, (iii) be structurally reconfigured to change the environment of an organism during its development, and (iv) generate precisely controlled heterogeneities (i.e., solid barriers, air pockets, chemical and soil biota gradients) in an otherwise homogeneous growing medium. This manuscript also proposes a broader concept: the use of reusable and mechanically interlocking building blocks for the construction of biological environments for cm-scale organisms and systems of organisms. Modular and reusable building blocks can alleviate the challenges associated with the large scales of plant science experiments, while providing new capabilities (e.g., controlled heterogeneities, reconfigurable environments) for the study of environmental effects on biosystem development. Furthermore, this concept provides materials chemists and engineers with two stimulating opportunities: (i) to creatively engage with the synthesis or development of increasingly capable cm-scale biological environments for important organisms such as plants, and (ii) to use these environments to test hypothesis concerning plants that are compatible with their skillset. Compelling opportunities lie in extending our approach to chemically synthesized bricks, LEGO-compatible 3D-printed bricks and objects, and commercial bricks from other manufacturers. Our laboratory will be introducing a set of integrated tools for the fabrication of frugal but sophisticated[37] cm-scale environments for the study of plants and other organisms[35].

Acknowledgments We thank Dr. Kuloth V. Shajesh for valuable discussions and William Rekemeyer for help in the laboratory.

Author Contributions Conceived and designed the experiments: LC. Performed the experiments: KRL TS SB AM. Analyzed the data: TS LC. Contributed reagents/materials/analysis tools: LC. Contributed to the writing of the manuscript: LC TS SB KRL.