Design - Build - Test - Learn
The goal of RareCycle is to bring an efficient, sustainable, and highly accessible e-waste recycling solution into the lives of communities all around the globe. Ambitious goals like this need structured and reliable engineering planning methods to become tractable. We used system decomposition to indentify the main constituent subcomponents of our overall goal, and structured our workflows accordingly. Then, we identified engineering cycles within our workflows and planned our work packages along the paradigm of Design-Build-Test-Learn.
System Decomposition
We decomposed our overall goal into three different subdimensions and their interactions. In doing so, we have created three subgoals that can be approached separately while also making sure the necessary information flows across subgoal lines are considered. Thinking like engineers, we defined specifications for our subgoals and surveyed methods that could help us achieve them from a bird’s eye view:
1. The biological heart of our project is to develop a biomaterial that employs metal-binding peptides on its surface. As mycelium is a promising candidate material, the primary goal is to engineer a living organism as close to a filamentous fungus as possible to express the desired properties. Of course, the peptides should selectively bind (groups of) Rare-Earth Elements. Building on the achievements of past iGEM teams 1,2 and other experts in the field, we want to contribute to a growing suite of these binding peptides. As any extraction process is preceded by a bioleaching of the e-waste, the extraction should be effective under acidic conditions.
Fig 1. | System decomposition with information flows.
2. To give our recycling process a physical hull, we design and build an apparatus that enables us to go beyond lab-scale and test our concept close to real life. As such, the apparatus should be a suitable, controllable environment for the growth of our fungal biomaterial, and possess the necessary functions to deploy it as a filter for bioleached e-waste solutions. The apparatus itself should be made from easily accessible and recycled materials and fitted to its future user’s needs. We used rapid prototyping to get a feel for the dimensions and potential issues in deploying our apparatus.
3. Introducing our idea to all stakeholders and incorporating their feedback is key to translating our project into real life. Therefore, we reached out to numerous industrial and non-governmental organizations to share our ideas and receive feedback. International connections played an essential role in tackling the global problem of e-waste recycling and helped us to question our own biases and fit our biotechnology to the needs of its future users. Therefore, we chose interviews as our principal method to obtain and share knowledge with our stakeholders.
Parallelized Workflows
Fig 2. | Parallelized workflows.
Based on the above specifications, we developed workflows that could be largely executed in parallel. On the wetlab side, we set up one group to work on producing and testing the capabilities of our Rare-Earth binding peptides. Another lab group dealed with the surface expression of an exemplary metal binding protein in S. cerevisiae as a stepping stone toward the surface expression in filamentous fungi. The Modeling subgroup ran simulations for the Peptide Lab on the one hand, and was a connecting bridge to the Hardware project in modeling the process conditions of our bioreactor/extraction apparatus. The Hardware group built the apparatus and - in parallel - worked on the suite of sensors to measure and control the process. Meanwhile, the Human Practices group conducted expert interviews and outreach programs. We see their activities also as an integral part of engineering, as their connections fostered user integration and guided our design principles with the Sustainable Development Goals.
Design - Build - Test - Learn Cycles
At the core of the technical workflows, we oriented ourselves by the Design – Build – Test – Learn Cycle. This cycle serves as a central guide for simplifying complex engineered processes, promoting transparency in engineered decision-making, and ensuring reproducibility. To illustrate its application in our project, three threads of Design – Build – Test – Learn - Cycles are now presented. We want to emphasize that although all cycles can operate independently, they are designed to complement each other.
Fig. 3 | Interconnected engineering cycles.
Hardware Design-Build-Test-Learn
Design
The principal design phase of our hardware project started with the idea of combining the growth of the fungal biofilter material and the Rare Earth extraction process within one apparatus. After brainstorming rough process sketches, we collected a list of requirements that should be fulfilled by our first design.
Fig 4. | Hardware requirement list at the design stage.
Then, we developed a CAD model to further guide our design and construction steps.
Fig 5. | CAD Model of the MycoFlux apparatus.
The CAD model went through a few iterations based on our literature research and expert and user feedback. Please have a look at our Hardware page for more information! As an engineering example, let’s examine the design process of one central building block of our device: the cultivation drawers. We started with the idea of using one large drawer to cultivate as much biomass as geometrically possible. Here, we were inspired by mycofiltration blocks which are used to remove contaminants from bodies of water like small lakes. On the other hand, we researched solid-state fermentation, used for example for the fungal fermentation of rice and soy beans. That technology often uses separate drawers to ensure the necessary air flow and oxygen supply for optimal growth and temperature control. To make a later evaluation possible, we adapted our design to incorporate both modes of operation as can be seen in the CAD model.
Build
It was very important to us to have a functioning prototype quickly, so we could gather user feedback and optimize our design. We built the apparatus to support both operation modes (single and triple drawers), so that both versions could be evaluated and compared. The drawers essentially consist of a wooden frame, lined with sheets of PTFE foil, and a mesh to support the biomass while letting filtered liquid pass through to the drawers or sump below. Learn more about our building process on our Hardware page. If you are interested in replicating our designs, consult our tutorial!
Test
To assess and study the cultivation drawers design and functionality, we devised different field tests.
1. Different metallic meshes (coarse/fine netting)
We found coarser metallic meshes to be more readily available and less expensive than finer ones. In the spirit of making our hardware low-barriers and easily accessible, we tested if a fine mesh is necessary or could be replaced by the cheaper option. By filling growth medium in two drawers, one equipped with coarse mesh and one equipped with finer mesh, and cultivating a wild-type filamentous fungi, we tested the retainment of biomass of the netting. Then, we did a test-run of our liquid distribution system using water and evaluated the liquid flow through the meshes.
2. Single / triple drawer operation mode
As mentioned above, we constructed both a large, single drawer and three smaller drawers out of inspirations from different existing technologies. We filled the larger drawer with cultivation medium and inoculated them simultaneously to the smaller drawers from the first test. We examined the biomass growth at regular time intervals and tested the throughput of water in a test-run of the liquid system.
Learn
From our tests we determined two main insights into the design of our cultivation drawers:
1. A finer mesh is beneficial for the liquid distribution to the next layer of biofilter below and is more successful in retaining solids that would otherwise have to be filtered out later with more effort. The economic impact of the different design choices is low enough to continue using finer meshes going forward.
2. The biomass is growing more evenly in the smaller drawers, confirming the benefits in aeration of tray-based solid-state fermentation. As we want to achieve a high area of contact with the filtered liquid in our specific use case, we will continue to develop the operation mode with three smaller drawers, instead of one large one. However, we could also confirm that the apparatus is suitable to host and cultivate high amounts of biomass in one large drawer, albeit slower, for use in other applications such as water filtration.
What we have learned in this first cycle is that the strategic-technical orientation of our apparatus is best suited to a tray-based system. Through these field tests we could also confirm the best placement of sensors and pipes in our final design.
Besides the cultivation drawers, we also cycled through designing, building, testing and learning for the liquid handling, sensor deployment, growth medium and general handling. We invite you to read our Hardware page for more information!
Peptide Lab Design-Build-Test-Learn
Design
The first step to develop a biomaterial that binds REE was to start the research on lanthanide binding peptides. We selected neodymium as an exemplary lanthanide that we wanted to extract from a bioleached solution of electronic waste. Then, we selected peptide sequences that could be feasible at bioleaching conditions and suitable to capture neodymium ions. Simultaneously, we assessed immobilization techniques of the peptides required for the binding assay.
We designed the plasmid to be expressed in E. coli as it is a model organism. Later adjustments to a different host such as yeast or fungi were incorporated into the design of the plasmid.
Build
We used twostep overlap PCR to build our construct inserting our genes of interest into a plasmid to contain designated components like LCI, promoter, and a Strep-Tag. After that we verified the insertion through sequencing, executed transformation and selected successfully transformed cultures. Using our newly produced strains, we checked expression, scaled expression and purified produced peptides. Conducting our leaching experiments, we produced two leaching solutions. One of an e-waste industry sample obtained from STENA Recycling, and one from a NdFeB magnet
Test
The majority of the tests involve the Arsenazo III assay which is a chemical test to detect the concentration of metal ions within a sample. The Arsenazo III reagent reacts with the metal ions and causes a color reaction that is spectrophotometrically measurable. This assay we first derived from its original use for it to be applicable on microtiterplates.
One of our first questions – using a colorimetric assay for almost all of our measurements – was whether the peptide would influence absorbance. For this we conducted an experiment with our different peptides in solution.
For our experimental setup and designated process, employing peptides on a matrix of sorts, we evaluated how well our peptide was to be immobilized on a polystyrene microtiterplate as a stand in for a hydrophobic bead.
We also evaluated the selectivity of the peptides for neodymium in heterogenous solutions such as solutions containing known ions with somewhat similarity and very heterogenous and acidic solutions gained from acidic leaching of an industry sample.
Fig 6. | Microtiterplate containing Arsenazo III assay sample solutions.
For our proof of concept, we also had to answer the question of what neodymium concentration to expect from leaching an actual industry sample, and if it would even be feasible. For this we set up different leaching solutes at different pHs and quantified these using our derived AS III assay.
Central for our assessment was the binding capacity and affinity of the peptides to lanthanoids. For this we conducted extensive tests with different samples. Starting with a simple neodymium millipore solution to first quantify affinity of the peptides, we graduated to testing how much neodymium the peptides could extract from a previously quantified leaching solution of an industry e-waste sample.
Learn
We found that our insert was difficult to integrate into our designated vector, so we experimented with different PCR setups. Doing this we learned to use a higher fidelity polymerase for difficult inserts and opted for a twostep PCR which grants both primers their specific optimal annealing conditions allowing insertion and amplification of our construct after all.
We found that our peptides affect absorption at the assay characteristic wavelength of 650nm, but then only in a negligible amount. By obtaining this knowledge we learned to always add peptide containing/treated blanks for any relevant experiment to be able to eliminate variance in data due to the observed absorbance, by subtracting any given effect from the absorption.
We successfully adapted the already previously established Arsenazo III assay for use on microtiterplates.
Fig 7. | Arno working in the lab.
We learned that the produced peptides fused with an LCI anchor on a linker are easily immobilized on polystyrene since we did not observe absorbance saturation following an HRP-ELISA after immobilization tests with up to 100 μL of 10 μM peptide solution.
Having initial problems with the conceptualization of an experimental setup for affinity and selectivity experiments without interference of the AS III with the peptide-ion interaction – we arrived at a setup treating immobilized peptides with test solutions, and then quantifying the removed supernatants with AS III. This setup has proved successful.
We concluded that the peptides bind neodymium very selectively, even if somewhat similar cations such as trivalent iron are present. From very heterogenous leaching solution from NdFeB-magnets or leaching solutions with unknown composition, neodymium is recovered selectively and in high amounts.
We also showed that with lower pH more neodymium is mobilized from e-waste through acidic leaching, and that this neodymium mobilized has significant amounts able to be recovered with peptides. Doing this we learned that photoactive components in the leaching solution would interfere with the AS III due to reflection/absorbance at the characteristic assay wavelength of 650 nm, making directly quantifying the leaching solutes impossible. Yet, we were able to overcome this obstacle by finding a workaround to still apply the AS III assay and quantify the leaching solution.
In affinity testing, we were able to bind neodymium from test solutions in significant amounts up to 95%. From a previously quantified leaching solution produced from an actual industry e-waste sample, we were able to recover over two third of neodymium contained.
We learned to successfully implement our ideas for peptide and experiments in design and build our construct and analog strains, build our experimental setups, tackle our challenges and implement our findings along the way enabling us for optimization of our procedures through iteration. We invite you to read our laboratory page for more detail.
Yeast Lab Design-Build-Test-Learn
Design
We initiated our project with literature research focused on cell surface expression in the yeast S. cerevisiae EBY100. Followed by our research, we designed a specialized pYD1-LanM vector to facilitate the cell surface expression of Lanmodulin (LanM) in S. cerevisiae EBY100, using the pYD1 vector and cells obtained from Invitrogen.
Additionally, we designed a plan for biopanning experiments aimed at identifying peptide sequences capable of binding neodymium ions using the phage library Ph.D.TM-12 by New England Biolabs (NEB).
Build
Regarding the building aspect of the DBTL-Cycle, the LanM gene was inserted into the pYD1 vector through restriction and subsequent ligation methods. We also amplified the plasmid in E. coli to increase its yield. Then the transformation was carried out, introducing the constructed plasmid into baker's yeast S. cerevisiae.
To ensure the accuracy of genomic transformations in both bacteria and yeast, we conducted verification through PCR and agarose gel electrophoresis.
Fig 8. | Picking of transformed colonies for verification of transformation through sequencing.
Test
To verify the recombinant expression of LanM in yeast we used the SDS-PAGE. In order to test neodymium binding capabilities of the surface expression construct, we designed neodymium binding assays using yeast cultures expressing LanM on their surface and compared it to the adsorption of cells only containing the unmodified pYD1-vector. This was performed for different cell and neodymium concentrations. The neodymium binding capacities of the yeast were tested by incubating different neodymium concentrations in a defined number of cells for both the positive and the negative control.
Additionally, multiple rounds of biopanning experiments were conducted, and isolated positive clones were subjected to Sanger sequencing to reveal both the peptide and DNA sequences.
Learn
Lastly, we have reflected on the knowledge we obtained and asked the questions that are still left unanswered. We achieved successful cloning of LanM into pYD1 and its subsequent transformation into S. cerevisiae EBY100. The surface construct could not be detected in an SDS-PAGE for certain. However, we could prove the significantly increased binding of neodymium ions for cells expressing pYD1-LanM on their surface in contrast to those only containing the unmodified pYD1 vector. This suggests a possibly false procedure regarding the SDS-PAGEs. We determined a neodymium binding efficiency varying from 24% to 57%, for the highest neodymium concentration of 50 µM during incubation 0,064 g neodymium was bound per 1 g yeast dry weight.
Additionally, the inserted peptide sequences of neodymium binding phages were identified via Sanger sequencing. One positively identified insertion found the following amino acid sequence: VVGSSGSTIPFP. This sequence could in turn be tested according to our peptide lab protocols.