bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.165928; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Title: openPFGE: An open source and low cost pulsed-field gel electrophoresis equipment Authors: Diego Lagos-Susaetaa, Oriana Salazara, Juan A. Asenjoa Affiliations: aCentre for Biotechnology and Bioengineering, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile Contact email: [email protected] Abstract: DNA electrophoresis is a fundamental technique in molecular biology that allows the separation of DNA molecules up to ~50 Kbp. Pulsed-field gel electrophoresis [PFGE] is a variation of the conventional DNA electrophoresis technique that allows the separation of very large DNA molecules up to ~10 Mbp. PFGE equipment is very expensive and it becomes an access barrier to many laboratories. Also, just a few privative designs of the equipment are available and it becomes difficult for the community to improve or customize their functioning. Here, we provide an open source PFGE equipment capable of the separation of DNA molecules up to, at least, ~2 Mbp and at low cost: USD$850, about 3% of the price of typical commercial equipment. Keywords: Biotechnology, Molecular biology, DNA Electrophoresis, Low cost equipment, Open source equipment, Pulsed-Field Gel Electrophoresis [PFGE] Specifications table Hardware name openPFGE Subject area Chemistry and Biochemistry Hardware type Biological sample handling and preparation Open Source License Hardware: CERN Software: GPL Documentation: CC BY Cost of Hardware $850 Source File Repository Current development version at https://doi.org/10.5281/ zenodo.3727331. Older and newer versions at https://gitlab.com/diegusleik/openpfge bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.165928; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1.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. In general, few equipment - i.e. PCR thermocyclers (7) - have an open source version, limiting the development of improved/custom equipment, and the costs of such equipment tended to be very high, which imposes a high barrier for democratizing its usage in many academic labs. PFGE equipment is expensive and exclusively sold by two providers [Bio Rad, Analytik Jena], they cost around USD$30,000 and do not have an open source version. Commercial PFGE equipment control of the electric field reorientation are based on both electronic circuitry [CHEF, Bio Rad] and physical rotating electrodes [Rotaphor, Analytik Jena]. Open hardware and free and open source software [FOSS] provides tools for fabricating devices at lower costs and higher adaptability. Easy to use microcontrollers such as Arduino and manufacturing technologies such as 3D printers has enabled a diverse community of developers to start building new customized, low cost and open source devices. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.165928; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2.Hardware description Hardware and circuit design and construction General outline Here we describe openPFGE, an open source and low cost PFGE equipment. The equipment implements a rotating gel electrophoresis [RGE] over an uniform electric field [Figure 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. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Peltier Fan Buffer element exchange Electrode 0 - 180° Water Heat Peristaltic block sink pump Buffer cooling unit Servo motor Circuit agarose gel ● Controls cooling unit reorientation ● Controls servo motor Circuit ● Parameters and control over Fixed electric field Bluetooth ● LCD run parameters LCD Electrode + + + + + + + + + + + + + Electrophoresis chamber High voltage 12 [V] power source power source Fig 1. openPFGE equipment diagram. Main components of openPFGE hardware: A servo motor reorient the agarose gel position by rotating a gel tray in respect to a fixed electric field. Movement of the servo motor is controlled by a circuit whose parameters are determined via Bluetooth by an Android app. Circuit also controls a buffer cooling unit.*. *Some icons made by Freepick, Google, Eucalyp and Those Icons from www.flaticon.com. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.165928; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Electrophoresis chamber Peltier cooling system Peristaltic pump Circuit Servo motor Gel tray LCD High voltage power source 12 [V] power source Fig 2. openPFGE equipment picture. General view of the RGE system implemented, highlighting its main components. 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. As the servo motor that performs the movement of the agarose gel in this implementation operates in 180° angle, the position of the gel in respect to the electric field can be specified at any time of the run in a -90° / +90° position. A zig-zag pattern in the direction of the electric field is applied to obtain a net straight migration of DNA molecules [Figure 3]. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.165928; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Electrode 0 - 180° Servo motor agarose gel reorientation [Left] Pulsed periodic reorientation of DNA Fixed electric field [Right] Net DNA migration Electrode + + + + + + + + + + + + + Fig 3. Electric field reorientation in RGE. Diagram shows [Left] the static electric field provided by the electrophoresis chamber electrodes and how the rotation of the gel by the servo motor alters the orientation of the electric field through the agarose gel and [Right] a zig-zag pattern in the direction of the electric field is applied to obtain a net straight migration of DNA molecules 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 x 150 x 90 [mm] chamber and a 100 x 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 degrees, 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.