We designed and built hardwares for protein fermentation and performance testing.
SHIELD stands for Self-made High-performance Inexpensive E.coli culture Lab Device. Where there is an ARMOR, there is a SHIELD, but why?
Our project requires the production and extraction of relatively large quantities of protein for mechanical or gel property testing. While flask culture confirms protein expression, its yield remains too low to meet our experimental needs. Through consultations with experts, we learned that commercial fermenters are prohibitively expensive and involve optimization processes too complex for our team's current capabilities. Similarly, specialized high-density fermentation systems in laboratories present a steep learning curve we cannot overcome within our timeframe. Therefore, we decided to develop a DIY fermentation device that is relatively inexpensive, simple to operate, and capable of achieving significantly higher cell densities than flask culture.
During the summer vacation, we participated in the CCiC conference and exchanged ideas with several other iGEM teams. We discovered that many iGEM teams face similar challenges in protein production: flask cultures provide insufficient scale and low protein yield; laboratory fermenters, while appropriately scaled, require complex operation and steep learning curves. Moreover, most iGEM labs lack access to such fermentation equipment; commercial fermenters are often oversized and prohibitively expensive for lab-scale protein production needs.
After interviewing multiple aerospace experts, we recognized the extremely high cost associated with launching all materials produced on Earth into space. For long-duration or deep-space missions, in-situ resource utilization—producing necessary materials in orbit or on planetary surfaces—will become an urgent requirement. Designing and building a low-cost, compact, and reliable space-compatible fermentation device would provide valuable insights for future human space exploration.
Open-Source 3D Printable Fabrication Entirely 3D-printed structure using standard materials can minimize component cost and enabling easy replication by other iGEM teams.
Balanced Portability and Performance Design was optimized to achieve high culture yield without increasing size, weight, or cost.
Modular and Evolvable Design Modular design principle allows components to be reconfigured, upgraded, or customized for individual needs — from routine lab use to future adaptations for space biomanufacturing.
We used a 1L wide-mouth blue-cap bottle as the foundation of our DIY fermenter. We drilled holes in the cap and inserted multiple fittings and tubes for aeration, feeding, and sampling.
Figure 1 and 2: SHIELD v1.0
A controllable feeding system was designed, using an IV bag as the nutrient reservoir connected to the device via a flow valve for precise control of nutrient supply rate and timing.
We used a mini aquarium air pump (2 L/min intake) to supply the oxygen required for bacterial fermentation. We drilled a hole in the cap, glued in a Luer connector, inserted the air line through the cap, and extended it into the medium.
An alkali replenishment port (Luer connector → 2.4mm adapter) was glued to the drilled lid, linked to a syringe with 10ml 1M NaOH. Litmus reagent was added to the medium for visual pH monitoring—turning red when pH drops below 7-8 to indicate the need for alkali addition.
The entire device (modified lid + bottle + attached tubings) was autoclaved to eliminate surface contamination; needle-type filters were installed at each tube end to block external contaminants during sterilization and operation.
To initiate the fermentation process in our Fermentation Device V1.0, we poured 300 mL of Luria-Bertani (LB) medium into the fermentation vessel, along with 10% (v/v) litmus reagent. Separately, we prepared a 500 mL solution of double-concentrated LB medium, to which we added 10% glycerol and 0.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). This solution was loaded into an IV bag to serve as the feeding medium—providing additional carbon and nitrogen sources for bacterial growth while inducing the T7 promoter to drive the expression of the target product.
To ensure the uniform distribution of nutrients and oxygen throughout the culture, we placed a magnetic stirrer at the bottom of the fermentation vessel. This setup enabled continuous mixing of the culture, ensuring that the bacteria could fully access oxygen and nutrients.
We then inoculated the fermentation device with Escherichia coli (strain BL21, harboring the Pet30A plasmid) engineered to express yellow fluorescent protein (YFP). After 6 hours of cultivation, we began to infuse the prepared feeding medium. The fermentation process was concluded after all the feeding medium had been completely infused over approximately 12 hours.
Following the fermentation period, we measured the optical density (OD) of the culture to determine the bacterial cell density. The OD value obtained was 1.2, indicating moderate growth of the bacterial culture.
Additionally, we conducted fluorescence tests to assess the expression of yellow fluorescent protein (YFP). We used blue light as the excitation source to observe YFP fluorescence, which qualitatively confirmed that bright yellow fluorescence was produced. Unfortunately, after further rigorous comparison, we found that the culture medium itself also emitted strong yellow fluorescence. Although the test group tube on the right appeared slightly brighter, the difference was not very obvious.
Figure 3: Fermentation result (yellow fluorescence)
Figure 4: Fermentation result: On the left is the LB culture medium as negative control, and on the right is our bacterial culture (the culture after fermentation was only slightly brighter than the reference medium)
Through practice, we identified key issues with SHIELD v1.0:
We consulted Professor Boxiang Wang, CTO of Lingzhu Technology and a protein fermentation expert, who recommended:
We observed a commercial fermentation device at Beijing Normal University. Noting its strengths (reliable interfaces/pipelines, integrated pumps, high-pressure resistance, automated pH control) and drawbacks (high cost/learning curve, expensive/replacement-delayed pH sensors), we got our improvement plans.
Synthesizing experience and expert advice, we plan to:
To optimize our fermentation device, we first decided to search for research papers and replicate others' successful medium-to-high density shake flask culture protocols—aiming to clarify the nutrient and oxygen requirements of the bacteria. A paper from East China University of Science and Technology caught our attention: the authors described a shake-flask method for producing squid ring teeth protein and claim they reached an E. coli density of about OD 20 in those flasks.
This article provided two critical clues:
We purchased 1000 mL shake flasks with baffles at the bottom (the baffles cause the medium to collide repeatedly during shaking, mixing significant amounts of air into medium). The culture process was as follows:
We tested multiple fermentation temperatures and achieved considerable bacterial yield and protein expression (see the experiment section webpage for details).
Without medium supplementation or pH adjustment, induced at 37℃, the shake flask's OD value reached approximately 15—far exceeding the SHIELD v1.0.
Based on feedback and challenges identified during initial experiments, we have implemented significant improvements to our fermentation device. The provided schematic illustrates the key enhancements.
The new design features a dedicated interface for pH adjustment using syringes to introduce sodium hydroxide (NaOH), and a sampling port that allows sterile extraction of culture samples for OD600 measurements with disposable syringes. Both initial inoculation and feeding now utilize Terrific Broth (TB) medium, providing a consistent and nutrient-rich environment for bacterial growth.
Nutrient feeding has been enhanced through integration of a peristaltic pump, which delivers nutrients at a controlled rate to maintain stable and continuous supply of carbon and nitrogen sources without disrupting the culture.
The fermentation vessel lid has been redesigned for manufacturing via 3D printing technology, enabling precise customization. The fastening screws are also 3D printed, with some featuring hollow centers to accommodate glass tubes directly—eliminating the need for additional drilling or modifications.
To address sealing integrity compromised during sterilization in previous versions, we implemented an external threading design on the lid and incorporated rubber gaskets between the glass jar and screws, ensuring a tight seal to prevent leaks and maintain system sterility.
For oxygenation, we acquired a high-capacity air pump (18 L/min) coupled with a dedicated 50-mm air filter to supply ample sterile air, meeting bacterial oxygen demands during protein production. An air stone positioned at the base of the aeration tube generates fine bubbles, significantly improving dissolved oxygen levels in the medium.
In summary, our redesigned fermentation device integrates multiple innovative features: controlled nutrient/alkali feeding, sterile sampling, efficient oxygen dissolution, 3D-printed lids and screws, and silicone gaskets for reliable sealing. While manual operations minimize costs, additional interfaces allow future upgrades for automated monitoring.
Figure 5: Assembly schematic showing two brown glass tubes for sampling/IPTG-NaOH addition and two blue tubes for feeding/oxygen supply
We selected polylactic acid (PLA) for 3D printing the device lid due to its cost-effectiveness and accessibility, making it ideal for educational and experimental applications. After printing, the lid underwent autoclave sterilization in preparation for fermentation experiments.
Upon completing the sterilization cycle, we expected a fully sterile, ready-to-use lid. However, inspection revealed severe damage—the PLA lid had warped and cracked under the high temperature and pressure conditions, rendering it unusable.
Figures 6 & 7: Cracked PLA lid after autoclaving
Consulting technical resources, we found that PLA softens above 60°C and undergoes significant moisture-induced expansion, leading to cracking. We subsequently researched FDM 3D printing materials and identified PAHT-CF (carbon fiber-reinforced polyamide) as optimal for our application. This material combines thermal stability, high mechanical strength, and low moisture absorption, enabling it to withstand autoclave sterilization while maintaining structural integrity.
We fabricated a new 3D-printed lid using PAHT-CF material, optimizing both printing parameters and thread dimensions before initiating sterilization tests.
Figure 8: The fully assembled equipment placed in the autoclave for sterilization. Results confirmed the lid maintained structural integrity without deformation or cracking, validating our material selection.
Figure 9: Rubber gaskets installed between the glass tube and screws ensure tight sealing, preventing leaks and maintaining system sterility (schematic illustration only; not actual fermentation equipment).
Figure 10: Close-up of rubber gasket installation
Figure 11: Additional view of gasket installation
Figure 12: Final view of gasket installation
We filled the SHIELD v4.0 with 1L of TB medium, with 200mL of 5× concentrated TB medium as feed solution for efficient nutrient delivery. After assembling all components (3D-printed lid with sealing gaskets, peristaltic pump), the complete system underwent autoclave sterilization. Fermentation was initiated under controlled conditions post-sterilization, featuring an aeration system enhanced with an air stone for oxygen supply and antifoam agent to maintain dissolved oxygen capacity.
Engineered E. coli Pat28a (designed to express N-gamma protein fragment) was used as the production strain. When culture OD reached 5, we added IPTG to 0.5mM final concentration to induce protein expression while initiating continuous feeding via peristaltic pump. Culture pH was maintained at approximately 7.5 (optimal for growth/expression) using pH test strips, with NaOH adjustments every two hours based on syringe-sampled measurements.
To precisely monitor the growth kinetics of our engineered strain throughout fermentation, we measured optical density (OD) at hourly intervals. The data summarized in the following table provides detailed documentation of bacterial growth over time:
Figure 13: Comparison of SHEILD Fermentation OD600 and normal shake-flask.
Our fermentation device demonstrated robust protein production capability, achieving OD values four times higher than conventional shake flask cultures. During SHIELD 2.0 testing, we employed specialized baffled conical flasks with 10% fill ratio to enhance oxygen transfer, obtaining twice the cell density of traditional shake flasks. Remarkably, SHIELD 4.0 not only achieved higher final OD values than SHIELD 2.0, but also produced 1200mL of culture in a more compact setup. This demonstrates our system can produce 10-100 times more protein than traditional shake flask methods at low cost and minimal footprint, without requiring large conical flasks, shakers, or expensive laboratory fermenters.
Through further user feedback, we plan to integrate a heating sleeve to eliminate dependence on incubators. These results highlight SHIELD's potential to enhance experimental capabilities, support biotechnology education, and advance space exploration. The system's controlled, efficient protein production represents a crucial step toward making biomanufacturing more accessible and sustainable. Our goal is to develop devices that not only improve experimental methods but also facilitate space biomanufacturing and education. This project successfully validates the feasibility of efficient, environmentally conscious protein production both terrestrially and potentially in space environments, establishing a foundation for future innovations in the field.
For the early developmental versions, our team conducted repeated internal testing to identify design flaws and converge on a robust, sterilizable, and easy-to-operate DIY system.
After iterative improvements, we finished a working device reaching an OD600 of 20 after overnight culturing. We then presented to experts and potential users for feedback.
Department of Biological Sciences, Beijing Normal University
feedback pro
That silicone ring design is really clever — it tightens the glass tube firmly when pressed, but you can still pull it out easily when needed. Super practical!
I’m surprised it doesn’t need a magnetic stirrer. The bubbles alone keep the culture mixing smoothly — that really cuts down the complexity.
feedback con
improvement
Tongji University iGEM team
"I really like the Luer connectors with screw caps. They hook up to syringes easily and can be opened and closed many times without contamination."
"Hardly anyone in this field has tried using 3D printing for fermentation devices. If you share your design openly, it will be a great start for others to follow."
Figure 14: Feedback from the iGEM team members of Tongji University on our fermentation device
Second High School Attached to Beijing Normal University; iGEM PI
"It’s small and simple, perfect for showing fermentation in a classroom. Everything’s visible. Great for teaching!"
Link Spider Tech. Co., Shenzhen, China
“I didn’t expect it could push OD above 20 with more than a liter of culture — that’s impressive. From my experience, that’s almost the limit of what you can reach without oversize pressurized devices.”
“At first I was worried it would foam like crazy, but your protocol added 0.005% antifoam after sterilization and it worked perfectly — minimal foaming bubbles.”
Figure 15: Automated pH sensor too expensive for SHIELD; heater band is cost effective.
Team Captain, BWYA High School Team
"Really easy to use — even high school students can handle it without much lab experience."
Space Engineer, Dr. Yao; members of the “Lunar Palace 1” Team
“The PP bottle with a 3D-printed cap is such a smart idea. GL45 caps are way too small for all these tubes, and modifying glass bottles would be expensive. This design is cheap, flexible, and it can ferment over a liter of culture — that’s amazing. If you could make a 5-liter or 10-liter version, that would be even better.”
Status
Figure 16: Display of all the parts of the SHEILD
1. Assembly of Equipment: As demonstrated in our instructional video, begin by carefully assembling each component of the fermentation device. Ensure that all parts are correctly positioned and securely fastened.
2. Inoculation of the Main Fermentation Vessel: Fill the main fermentation vessel with 1 liter of Terrific Broth (TB) medium. Add the seed culture at a 5% ratio along with the appropriate antibiotics to select for your engineered E. coli strain.
3. Preparation of Feeding Solution: In a separate container, prepare the feeding solution by adding 200 mL of a 5x concentrated TB medium. This solution will provide additional nutrients to support bacterial growth during the fermentation process.
4. Incubation and Monitoring: Place the entire fermentation setup into a 37-degree Celsius constant temperature incubator. Set the oxygen flow rate to ensure adequate aeration. Connect the sampling port (Luer fitting) to a syringe and draw 2 mL of the culture every hour to measure the optical density (OD) and pH levels.
5. Induction and Feeding: Once the OD600 of the culture reaches 6, induce protein expression by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) through an additional Luer fitting. Begin the feeding process using the peristaltic pump at a rate of 0.5 mL/min to deliver the concentrated TB medium.
6. pH Adjustment: Monitor the pH of the culture closely. If the pH drops below the optimal range, adjust it by adding sodium hydroxide (NaOH) or ammonia solution through the Luer fitting. This step is crucial to maintain the physiological conditions necessary for protein production.
7. Fermentation Duration: Allow the fermentation to proceed for a total of 24 hours. During this period, continue to monitor and adjust the OD, pH, and feeding as necessary to ensure optimal growth and protein expression.
8. Harvesting: After 24 hours of fermentation, proceed with the harvesting of the bacterial culture. This step will involve separating the cells from the medium, typically through centrifugation, and preparing the sample for further analysis or processing.
In this study, we aim to investigate the properties of a specific protein under various environmental conditions. Through interviews with experts in the field, we have determined that key properties such as tensile and shear forces need to be measured. However, as a high school team, we face significant challenges in accessing professional equipment for these measurements. This limitation is not unique to our team; other high school teams participating in iGEM competitions also struggle to obtain the necessary equipment. Consequently, we have embarked on the development of a custom hardware solution to measure these properties. Our goal is not only to fulfill our own research needs but also to provide a feasible and innovative design that can serve as a reference for other iGEM teams facing similar challenges.
Figure 17: Testing device for protein tensile force
We have developed a device for measuring the tensile strength of proteins, constructed primarily from LEGO EV3 materials, which is cost-effective and affordable for replication by other high school teams. The blue component at the base of the device is a force sensor. The experimental setup involves placing two adhesive blocks connected by a protein with adhesive properties between the sensor and a motor-driven block that moves upward. The other block remains stationary and is held in place at the bottom. The force sensor measures the tensile force exerted by the protein as the two blocks are pulled apart by the motor. The measurement continues until the two blocks connected by the protein are completely separated, at which point the tensile strength of the protein is determined. To measure the tensile strength of proteins under different temperature conditions, the blocks are simply placed in a refrigerator or an oven for one hour to acclimate to the desired temperature before conducting the experiment.
The assembly process is straightforward, requiring no specialized tools or expertise beyond basic mechanical understanding. This makes our device an ideal project for educational purposes, as it encourages hands-on learning and fosters an understanding of the principles behind protein tensile strength measurements. By sharing our design, we aim to empower other teams to conduct similar experiments, thereby promoting scientific inquiry and collaboration among high school students interested in synthetic biology and material science.
Concurrently, we have designed a device to measure the rate at which a protein forms a hydrogel. This device is composed of two parts that can be tightly secured together, with the outer casing 3D printed from PLA material. After printing, the casing is coated with a layer of resin to ensure its impermeability. Inside, we have placed a disc made from a material that simulates the composition of a spacesuit (a blend of acrylic and mouse pad material), which has been drilled with a small hole. This setup is used to assess the protein's sealing ability by applying the protein to the hole and monitoring the time it takes for the protein to form a hydrogel that seals the opening. Additionally, the device can be placed in a vacuum environment (by sealing it in a container and evacuating the air) to observe how the protein behaves under vacuum conditions. This device is simple to construct, requiring only access to 3D printing technology, and is capable of measuring the protein's gelation process, making it an ideal tool for high school iGEM teams to use.
Figure 18-20: The device for testing the ability of proteins to form hydrogels