Team:Lambert GA/Excellence
OVERVIEW
OpenCellX, a continuation of Lambert iGEM’s hardware project from 2019, is a viable, frugal substitute for some of the most common lab devices used in synthetic biology labs - namely, benchtop centrifuges, vortex mixers, and tissue homogenizers. Using off-the-shelf components, including an Arduino microcontroller, brushless motors, LCD display, 3D-printed components, and a modular 3D-printed chassis, the total cost for OpenCellX is just $65. Current testing has shown that OpenCellX is able to produce comparable or even matching results when tested against commercial lab-grade equipment. These devices are an integral part of many synthetic biology workflows, including the bacterial plasmid minipreps that the 2020 Lambert iGEM team performed for AgroSENSE. With a focus on frugality, efficiency, and versatility, OpenCellX has evolved into a machine applicable to various workflows and a variety of environments, truly making synthetic biology more accessible for all.
DESIGN
HOMOGENIZATION MODULE THEORY
Figure 1. The Benchmark Scientific BeadBug, a commercial Bead-Homogenizer
A commercial tissue homogenizer creates high frequency oscillations, shaking a tube and all of its contents vigorously to lyse tissues and cells. The most common method to achieve such high oscillations is a motor, in conjunction with a heavy spring, to shake tubes along an arc. This mechanism, although effective, requires a very powerful motor, thus creating unnecessary expense.
Figure 2. A render of the 2019 OpenCellX design, as presented at the 2019 Giant Jamboree.
Figure 3. A High Speed Recording of the previous OpenCellX design at 1502 FPS, taken at the BhamlaLab.
Inspired by the motion of a bike pedal and its hyper efficient transfer of linear motion to rotational motion, the 2019 Lambert iGEM team designed a homogenization mechanism using an epicyclic planetary gearbox. As the device revolves around a fixed central gear, secondary gears turn in an opposite direction to the motion. A final set of tertiary gears, which are connected to the tubes, are driven by the secondary gears. This unique combination of gearing, both internal and external, creates a system where the tubes and larger mechanism revolve around the center axis, while a counter rotation caused by the internal gearing cancels the relative rotation about the tubes’ central axis. The end result is a mechanism in which the tubes maintain their relative orientation, always pointing towards the same direction as they revolve.
Within a tube itself, the unique kinematics take advantage of the centrifugal acceleration caused by the high-speed revolution to create oscillations along a two-dimensional plane. The centrifugal forces are constantly pushing the contents inside the tubes outwards, radially, from the center of rotation. However, as the orientation of the tubes, relative to the direction of the centrifugal force, are constantly changing. For example, at the beginning of the cycle, the centrifugal force facing radially away from the center of the mechanism points towards the cap of the tube, pushing the contents towards the cap. After a 90 degree revolution, the same force is now pointing towards the side walls of the tube, pushing the contents of the tube to the side. Finally, after a 180 degree revolution, the force is now pointing towards the bottom of the tube, pushing all of the contents to the bottom. As the cycle repeats, the centrifugal forces push the contents of the tubes up and down the length of the tube, thus creating oscillations.
Figure 4. A High Speed recording, taken at 960FPS, of the improved OpenCellX design
Figure 5. The arrow represents the centrifugal acceleration. The sample is in dark green.
With a relatively light rotating mass, a continuous and even cycle, and no heavy springs, OpenCellX is able to efficiently convert the rotational motion from a motor into rapid linear oscillation within sample tubes, all while requiring less power and being cheaper than traditional tissue homogenizers.
Figure 6. Using OpenCellX to vortex a solution of food coloring, water, and corn starch. Shown at 960FPS
Furthermore, because the unique kinematics of the system are constantly shaking the contents within a tube, the OpenCellX homogenization module also functions as a vortex mixer.
CENTRIFUGATION MODULE
The OpenCellX Centrifugation Module is much simpler in design and mechanics than the Homogenization Module. Because the brushless motor is already able to provide rotational energy at very high RPMs, the goal of the main design considerations was to reduce weight and size to optimally use the motor’s energy. Just like the Homogenization Module, it can be quickly replaced with another module just by removing the locking nut. At the base of the module, two small magnets are attached so that the software can measure the precise RPM - and by extension, RCF - at all times.
Figure 7. OpenCellX with the Centrifugation Module attached.
Initial testing for this attachment was performed using samples of cornstarch, water, and red food coloring as a proof of concept. At a sustained 1500RCF, the module was able to pellet out any cornstarch in the sample after just 30 seconds. Satisfied with these results, Lambert iGEM continued testing by pelleting out homogenized samples of spinach, pelleting out cells from a liquid culture, and by passing samples through spin columns. The positive results showed that the lower RCFs were sufficient for a full DNA extraction and miniprep workflow.
Figure 8. 3 samples of a mixture of water, cornstarch, and red food coloring. Centrifuged using OpenCellX for 30 seconds at 1500RCF
Figure 9. One of 5 spin columns processed using the OpenCellX Centrifugation Module at 200RCF for 5 minutes.
Figure 10. A homogenized sample pelleted using the OpenCellX Centrifugation Module at 200RCF for 5 minutes
Figure 11. A triplicate of liquid cultures of bacteria processed using the OpenCellX Centrifugation Module at 2000RCF for 5 minutes.
IMPROVEMENTS
ELECTRONICS AND PROGRAMMING
Previous designs of OpenCellX were prone to a few issues: inconsistent speed, excessive vibrations, and stress related fractures in 3D-printed components. To address these, OpenCellX has been redesigned to utilize ball bearings at every rotating component, reducing vibrations and wear significantly. To ensure consistent operation, a hall-effect sensor, which detects magnetic fields, was added into the design to detect the presence of magnets incorporated into the rotating mechanism. The Arduino microcontroller then measures the time between detections to determine the rotational velocity of the mechanism, measured as RPM. With the ability to measure RPM, speed control algorithms have been added to the OpenCellX code to maintain an RPM of 2% of the desired value.
Figure 12. Electrical Diagram of OpenCellX.
MECHANICAL DESIGN
The next improvement to the OpenCellX system is a redesign of the gear train so that the motor can be placed at the center of the mechanism. In the previous OpenCellX design, the motor was placed off to the side of the mechanism, powering the mechanism through external gearing. This was done to reduce the load on the motor and simplify the design of the internal mechanism. However, doing so significantly limited the maximum RPM of the system itself to an experimental 3000RPM without load, resulting in poor centrifugation performance as the device was unable to generate enough Relative Centrifugal Force (RCF) and limited the configurability of the project itself. To create a more modular device, where centrifugation and homogenization attachments can be swapped easily, the DC motor from the previous design was replaced with a more powerful brushless motor, and the motor was shifted to the heart of the mechanism itself. With this change, switching between centrifugation and homogenization modes is as simple as removing the locking nut and replacing the modular attachments. Furthermore, the new motor placement significantly increases the maximum RPM that OpenCellX is able to sustain, from 3000RPM to over 5000RPM under load.
Figure 13. The previous OpenCellX gear train, the motor can be seen underneath the orange external gear.
Figure 14. The updated homogenization module, the motor is enclosed within the central gear.
DEVELOPMENT WORKFLOW
OVERVIEW
The developmental process of the OpenCellX project has remained largely unchanged from last year’s project. Lambert iGEM’s hardware team utilizes the Engineering Design process, a cycle involving gathering feedback from the intended user, researching ideal methods to achieve the desired result, using CAD design software to virtually design devices, creating prototypes using 3D printing technology and electronics breadboarding, testing and evaluating the prototype within its intended use-case, and seeing what components warrant improvement.
SOFTWARE
The Lambert iGEM team utilizes a variety of software throughout its workflow, starting with Autodesk Fusion 360, a highly functional CAD design software. Fusion 360 is how the hardware committee creates 3D models off all of the components that compose its various hardware devices. Here, the team can determine the optimal arrangement of components and simulate certain conditions to evaluate the functionality of designs. The cloud integration and collaborative features are unique, enabling Lambert iGEM to work more efficiently. Fusion 360 is able to output 3D models in the form of .STL files, which are accepted by the team’s slicer of choice, Ultimaker Cura. This software converts 3D models into instructions that a 3D printer can understand, called GCODE. With Cura, Lambert iGEM can also adjust an array of different parameters that will affect the quality, strength, and weight of 3D printed components. On the electronics side of the workflow, the team uses Fritzing, a circuit diagram tool, to design the circuits that connect all the different sensors, microcontrollers, and electronics that comprise the team’s projects. Finally, to program microcontrollers, Lambert iGEM utilizes the Arduino IDE, an integrated development environment capable of compiling C/C++ code into instructions that Arduino based microcontrollers use to operate.
RESULTS
SPINACH
As with the 2019 project, Lambert iGEM began testing of the new OpenCellX system by performing a DNA extraction workflow with the Commercial Qiagen DNEasy Plant Pro Kit and comparing the results to a commercially available tissue-homogenizer. With the help of the Bhamla Lab at the Georgia Institute of Technology, Lambert iGEM was able to source the Benchmark Scientific BeadBug Benchtop Homogenizer as a comparison.
Figure 15. DNA Yield results compared between OpenCellX, operating at 750RPM, and the BeadBug operating at 300HZ. both were run for 120 Seconds.
This figure shows the resultant DNA yields, in µg of DNA, after processing triplicate samples of spinach for 120 seconds on both machines at 75% speed. Although the OpenCellX yields are marginally lower, Lambert iGEM’s experience indicates that any values above 2.5 µg are more than sufficient for downstream applications. As data showed acceptable results, Lambert iGEM concluded that the changes made to the OpenCellX design were not detrimental, and continued testing by adjusting the processing time.
Figure 16.The DNA Yield results, as measured by a Nanodrop, after processing samples at different time invervals at 750RPM
Figure 17. The DNA Purity results, as measured by a Nanodrop, after processing samples at different time invervals at 750RPM.
The team processed all samples at 750 RPM, as higher speeds had a high risk of shattering the tubes supplied by the DNEasy Plant Pro Kit. Using OpenCellX as the primary vortex mixer, and a commercial centrifuge for later steps, there was a clear trend between the results and processing times. It was apparent that a longer homogenization time improved both DNA yields and consistency of the team’s results, as can be seen by the smaller Standard Error Measurement (SEM) error bars. For DNA purities, which are measured as the A260/A280 ratio from a nanodrop, longer homogenization time produced better results, but not by a significant or even noticeable margin. With a purity of 1.8 as the accepted value for pure genomic DNA, and 2.5 µg as the team’s threshold for acceptable DNA yield, Lambert iGEM concluded that a 60 second run on the new design was sufficient for most DNA applications. These results, showing good yields and purity, also prove that the device has functional vortexing capabilities.
Figure 18. The DNA Yield results of the improved OpenCellX Design operating at 750RPM as compared with the previous design at max speed. No data points for 2020 OpenCellX were taken at Homogenization Time greater than 300 seconds
Comparing these results to a similar trial from the previous OpenCellX design, the effects of the improvements in efficiency and consistency are evident. Not only are the values for the newer design much higher - they are also much more consistent at almost any time interval.
Satisfied with the team’s plant testing, Lambert iGEM is currently shifting to testing OpenCellX in bacterial workflows. Although there is not significant need for tissue-homogenizers, centrifugation is a key process in most bacterial workflows.
Figure 19. The DNA Yield results of the OpenCellX Centrifugation module, operating at 2800RCF as compared to a benchtop centrifuge.
Although this was a preliminary test, the data shows promising results. The benchtop centrifuge was operated at 12000RCF, as called for by MiniPrep protocol. OpenCellX was run at 2000RCF; to account for the significantly lower RCF, the team added an additional 5 minutes to each centrifugation step when performed on OpenCellX. Biological replicates were processed from the same 50.0 milligram amount of lettuce.
REFERENCES
[1] Benchmark Scientific. (2019, December 20). BeadBug™ 3 Position Bead Homogenizer. Retrieved October 28, 2020, from https://www.benchmarkscientific.com/rpproducts/bead-bug-3/