openPFGE: An open source and low cost pulsed-field gel electrophoresis equipment

Graphical abstract


Hardware in context
DNA electrophoresis is a frequently used technique in molecular biology that allows the separation of DNA molecules up to~50 Kbp by applying a single-direction electric field across a slab of agarose gel where small DNA molecules are sieved in a size-dependent manner. However, the sieving effect underlying such separations fails when very large DNA molecules must be resolved, as is the case of genomic analysis using intact chromosomal DNA molecules or analysis and manipulation of very large restriction fragments. Pulsed-field gel electrophoresis [PFGE], developed by Schwartz and Cantor [1], is a variation of the conventional DNA electrophoresis technique that allows the separation of very large DNA molecules up to~10 Mbp. This is achieved by a periodic and abrupt change in the direction of the electric field that exploits the reptation phenomena of large DNA molecules to allow size-dependent electrophoretic mobility.
Bacterial subtyping is the main PFGE application due to the discriminatory power, simplicity and low cost of the technique. Although some laboratories are transitioning towards whole genome sequencing-based typing, it remains important for small hospitals and laboratories with limited resources and it will be the more feasible option for bacterial subtyping by longer [2,3]. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments [4] as is the case, for example, of Cas9-assisted targeting of chromosome segments [CATCH] [5] or DNA fragment size assessment for Next-Gen Sequencing [6].
Biochemistry is an area where highly sophisticated equipment is required.  1 & 2], using the basis of the system described by Southern in 1987 [8]. The design considers the use of a standard commercially available electrophoresis chamber and the construction of an agarose gel rotation system using 3D printed parts, a servo motor and simple electronics. It incorporates a custom cooling system using a small pump and peltier cooler elements. The control of the motor, the cooling system and the parameters of the run are made via bluetooth communication to a smartphone running an Android app.

RGE system
In a RGE, the electrodes of the electrophoresis chamber remains fixed while the agarose gel is rotated to achieve the reorientation of the direction of the electric field across the gel.
Angle and switch time are a critical parameters in achieving optimal resolution. Separations of larger sizes of DNA are greatly improved by the use of a smaller angles. Using smaller angles results in savings in run time (up to 50%) [9]. openPFGE receives fixed angle as a parameter of the run and can take values from 0°to 180°. A zig-zag pattern in the direction of the electric field across the agarose gel is applied to obtain a net straight migration of DNA molecules [ Fig. 3].
In PFGE, the switch time is defined as the length of time the electrical field is applied on each direction of the zig-zag pattern. Some PFGE protocols require fixed switch time while others (samples with wide range of DNA fragment sizes) require changing of the switch time over the course of the run. This is referred to as switch time ramp. This ramp is specified by three parameters: an initial and a final switch time and a total electrophoresis run time. A linear or a nonlinear progression between these values during the electrophoresis is then performed. In openPFGE, fixed switch time and linear switch time ramp modes are available. The servo motor moves the agarose gel from one direction to the other at top speed after switch time is achieved on a single direction.
In order to keep the design simple and the cost as low as possible, a gel rotating system inside a standard large electrophoresis chamber was implemented in this design. In this case, a 310 Â 150 Â 90 [mm] chamber and a 100 Â 60 [mm] agarose gel is proposed, but it can be adapted to any chamber/gel size. This gel rotating system incorporates simple 3D printed parts composed of: 1) a tray base, that supports the gel; 2) a tray cover, that fixes the gel position; 3) a stem, that goes from the tray cover through the electrophoresis chamber cover and joins the servo motor; and, 4) a joint, of the stem to the servo motor that allows easy release of the tray using a strong magnetic couple. It also includes a 180°, 5 [V], high speed and accuracy digital servo motor -like the DS3218-MG -that allows the PFGE to move at any specified angle. All 3D printed parts were designed considering the Fused Deposition Modeling [FDM] process by using mostly horizontal and vertical faces, low inclination angles and no -or really small -ceilings, therefore, no support structures are needed. Fig. 4 shows the gel tray and servo joint design.

Cooling system
The cooling system is composed of a small 12 [V] peristaltic pump that circulates the buffer through a peltier cooler element based refrigeration system consisting of two peltier 12 [V]/5 [A] elements, an aluminum heat dispenser and two 12 [V] fans [ Fig. 5]. An epoxy covered NTC thermistor allows to sense the current buffer temperature in order to define the start/ stop of the cooling system.

Electronic components
The circuit is driven by an Arduino Nano microprocessor. It is supplied by a 12 [V]/30 [A] power supply and has a 5 [V] regulator (LM7805) to provide power to the microprocessor, components and servo motor. It uses a HC-05 Bluetooth module to provide communication to a smartphone for system control. It holds a serial LCD/I2C module to display the parameters of the electrophoresis run independent from the smartphone. The NTC thermistor signal is read by an analog input and peltier elements, fans and the pump are controlled by digital outputs through high current MOSFETs. The PCB design can be found at the Gitlab repository [10] or Zenodo repository [11].
The electrodes of the electrophoresis chamber are connected to a standard commercial electrophoresis high voltage (HV) power supply. It has to be manually operated as the circuit of the openPFGE does not have, yet, control control over its function.

Firmware
The Arduino Nano microprocessor runs a firmware that allows the control of the components. The Arduino control motor rotation. It reads the analog signal from the thermistor and activates the refrigeration system when the temperature exceeds set values. The Arduino sends current parameters to the LCD/I2C module display. Finally, the Arduino communicates with the Android app by bluetooth for setting parameters and monitoring the device. It allows the run to have a fixed switch time or a switch time ramp. Switch time ramp consists of three parameters: start run time, end run time and run duration. A linear progression of switch time ramp is calculated based on these parameters. The firmware allows the equipment to run normal PFGE. Other run modes (as FIGE) are expected to be available in the next firmware/app update.

Android app
A dedicated android app was developed in order to control the equipment. It establishes communication with the PFGE via Bluetooth and capture user preferences as: on/off, pause, switch time ramp on/off, ramp start time, ramp end time, ramp duration time, wait on position time (fixed switch time), rotation angle, total run time, buffer temperature (setpoint), temperature automatic control on/off, among other options. Is has the option of load predefined run protocols for commercial markers as well to save user defined programs. Fig. 6 shows the main views of the app. App download is available at Google play store [12].

Repository and open source hardware certification
All 3D parts design files, code and electric diagrams can be found at the Gitlab repository [10] or Zenodo repository [11]. The equipment has been certified under OSHWA certification: [OSHW] CL000001 | Certified open source hardware | oshwa.org/cert.

Table in
Bill of materials section summarizes the costs of each component of the equipment, including electrophoresis chamber and HV power supply. Commercial PFGE equipment is around USD$30,000. The total cost of openPFGE equipment is about USD$850, therefore it is about 3% of the price of a typical commercial equipment, considering electrophoresis HV power supply and chamber. Of this cost, around USD$720 correspond to electrophoresis HV (USD$350) power supply and chamber (USD$370), which many labs have already on hand. Then, the cost of the remaining components of this project are just USD$130.

Discussion
In order to contribute to the democratization and acceleration of hardware development in biochemistry, we present in this work a certified open source hardware that implements rotating gel electrophoresis to perform PFGE. All 3D parts design files, code and electric diagrams are open source and publicly available at the Gitlab repository [10] or Zenodo repository [11]. The equipment is capable of the separation of DNA molecules up to, at least,~2 Mbp, costs about USD$850, about 3% of the price of typical commercial equipment considering the chamber and the HV power supply. OpenPFGE has similar requirements, capabilities and results than commercial equipment in terms of applications, maximum fragment size resolution and experiment run time/setup. As it is the first version of the equipment, further improves in design of components, electronics and software can lead to a more robust implementation. This, together with validation of building and use by  other research groups could end in a equipment with similar application confidence than commercial equipment. Although open blueprints for PFGE instrument has been described earlier [13], to our knowledge, this is the first PFGE fully documented, ready to build, open source, flexible and modern equipment available.
Other modes of use such as field inversion gel electrophoresis [FIGE] are intended to be added in the future among other applications like, for example, DNA size selection for Next-Gen Sequencing. Open source versions of a gel electrophoresis chamber [14] and electrophoresis HV power supply [15] are publicly available. Is expected to develop versions of these components to fit openPFGE in order to bring a whole open source PFGE equipment, with both lower costs and flexible design.
The openPFGE

Software
Arduino IDE software was utilized for the microcontroller programming. Android studio was utilized for the app development, using API 29 (Android 10) and min API 26 (Android 8). The use of each of the design files is best determined by the descriptions and rendering shown in Build instructions section.  1 Power supply: The 12 [V] power supply input is connected to high voltage. Make sure this connection is properly isolated and without risks of water/buffer dropping. Be sure the power supply has enough fresh circulating air in order to eliminate the heat produced by the circuit. 2 Circuit: The circuit is powered by 12 [V]. This may not be considered dangerous for the operator. Make sure this circuit is properly isolated and without risks of water/buffer dropping in order to prevent circuit damage. Be sure the circuit has enough fresh circulating air in order to eliminate the heat produced by the circuit.

To make a PFGE run:
1) Download and install the openPFGE Android app from Google Play Store [12] 2) Load your agarose gel with the samples on the gel tray. Close the gel tray using the pins 3) Introduce the tray to the electrophoresis chamber and connect the tray to the motor join and close the electrophoresis chamber. Make sure to level the gel tray about 1[cm] above the bottom of the chamber using the screw provided for this. 4) Add the appropriated electrophoresis buffer to exceed the top of the gel tray in about 1[cm] 5) Power on the 12 V transformer of the circuit 6) Use the smartphone menu to pair Blue tooth to the openPFGE-*** module 7) Open the openPFGE app and configure the run parameters 8) Tap the send button on the menu bar and wait for the confirmation of successful received from device message 9) Power on and start the electrophoresis HV power supply. That's all

PFGE marker validation
Two commercial available markers were tested using openPFGE equipment under similar run conditions as the provider recommendations in terms of buffer, agarose concentration, ramp times and rotation angle. Fig. 10 and Fig. 11 shows the comparison between a commercial marker reference image and the result obtained with this equipment for the MidRange PFG Marker (Max size band~250kbp) and CHEF DNA Size Marker #1703605 (Max size band~2.2Mbp).

PFGE marker validation
In order to validate the operation of the equipment, two commercial markers were tested under the following conditions,

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.