1. Name of applicant(s)
The Friendzymes Contributors
- Jeremy Cahill, USA
- Isaac Larkin, USA
- Sarah Ware, USA
- Isaac Nuñez – FluoPi, Chile
- Scott Pownall, Canada
- Homer Sajonia II, Philippines
- Jacob Segarra, USA
Friendzymes Advisor
- Sebastian Eggert – OpenWorkstation, Germany
2. Email address (or preferred and reliable way of official contact)
3. What track are you applying to? (select one):
- Established Project Track
4. Tell us about your project in one or two sentences
Friendzymes’ goal for this project is to create a low cost open automated colony picking robot to facilitate molecular cloning of assembled DNA parts from Freegenes, iGEM, and other open wetware sources as well as for colony screening of strains for desired assemblies and behavior. This device can also be used in a wide variety of settings and purposes from isolating microbes through environmental sampling to screening of organisms for antibiotic activities.
5. Describe your project goals and how you expect to achieve them
Friendzymes’ mission is to democratize and globally distribute the means of biotechnological production. The goal of this project is to design and build a low cost cartesian-based open automated colony picking robot using the Raspberry Pi and associated camera system.
The first functional stage of the system will photograph microbial (bacteria and yeast) colonies on agar plates of differing form factors. Options will include light field and blue light fluorescent colony identification. Software and metric calibrant labware will identify the coordinates of isolated colonies from the images which could afterwards be selected manually or automatically for picking.
The second functional stage will employ a newly-developed picking head to pick isolated colonies using the information of imaged agar plates and transfer them to either another agar plate or 96-well deep dish or cluster culture tubes for growth. The system will be extensible by permitting a wide variety of transfer destination options and sterilization modalities.
6. Approximately how many people would be working on your project?
For our phase I, the core design team is made up of six researchers from the Philippines, US, Canada and Chile. The team has complementary expertise in software development, hardware design, and molecular biology. Other Friendzymes team members around the world are interested in collaborating.
7. Describe how your organization will create and manage collaboration with others.
Friendzymes operated as a gold medal winning unique team during the 2021 iGEM.org competition with team members from 9 countries and four wetlabs located in Ghana, the Philippines, the US and Canada. This allowed us to create a foundation for global communication. Our team hosted a weekend hackathon with participants from around the world including Indonesia. We had team members supporting the hackathon in most time zones. Friendzymes main tool for communication is via our Discord channel but we also are on JOGL’s Slack channel. Friendzymes also has its own public Github account where all details will be disseminated from. Friendzymes continues as a multi-focal project, with individual team members continuing to organize regularly scheduled team calls and thematic development sessions. This colony picker project will proceed in the same fashion.
8. Does your project have representation for a marginalized demographic due to factors such as race, ability, place of birth, gender, sexual orientation, socioeconomic class situation or other identification? If so, how?
We applicants have diversity in nationality, culture, ethnicity, gender and socioeconomic class, and include multiple members in low/middle-income countries.
9. What resources / infrastructure do you currently have to support your project?
Friendzymes has nonprofit status and a bank account through fiscal sponsorship by GOSH.
BioBlaze community lab is based in the Chicagoland area, and has direct access to lab equipment including an Opentrons OT-2 liquid handling robot, a MinION DNA sequencer, a thermal cycler, an orbital shaker, an incubator, and a centrifuge. BioBlaze is also nearby and can access laser cutting and FDM 3D printing services from third parties in the Chicagoland area.
The Philippines location is equipped with an electronics lab and 3D FDM printers. Local access to extra production capacity in machine shops for metalwork and public makerspaces such as FabLab Davao for 3D printing is also available.
Open Science Network’s community lab is BC (Canada)-incorporated non-profit society and is located at MakerLabs.com, western Canada’s premier makerspace. This gives us access to a wide range of maker tools including a full spectrum Laser cutter, 3D FDM printer, Tormach PCNC 1100 CNC Mill, metal laser cutter, metal lathe and electronics lab. The OSN community lab itself has a fully functioning wetlab that includes an Opentrons OT-2 with GEN2 P20 and P300 pipettes and consumables, Opentrons heat block and magnetic block modules, bacterial incubators including orbital shaker, thermocyclers and qPCR machines, a variety of centrifuges, pipettes and tips, protein & nucleic acids gel electrophoresis tanks and power supplies, sonicator, autoclave, -80 and -20 freezers and refrigerators, water & dry baths, magnetic hotplates, uv/vis spectrophotometer, ELISA Reader, a variety of optical microscopes, ductless chemical hood and laminar flow hoods.
10. What will you use the funds for? Describe your budget. List what you are going to spend it on and how.
For phase 1, funds will be used to purchase (and, as needed, internationally ship) the components require to construct, and modify as needed, open-source FluoPi fluorescence and epi-illumination imaging systems, including both raw materials and laser cutting & 3D-printing services; as well as to prototype and develop physical metric calibrants with the footprint of a standard microplate. Additional funds will be used to purchase lab reagents and plasticware, required for growing and plating E. coli cells to serve as test beds for colony imaging and picking. Finally, funds will be used to prototype subcomponents of a dedicated colony picking robot, including modules for sterilizing, cutting and/or washing a plastic filament or metal rod that does the picking.
11. How will you share the outcomes of your project? What documentation will you provide so that it will benefit the community as a whole?
(videos? photos? a how-to?)
Friendzymes has its own public Github account and repository has been created to document the progress as well as the final project files which can comprise of but will not be limited to:
- Final BOM
- Technical Drawings
- Behavior Documentation
- Firmware source code
- PAML Specialization Class
- Part STL Files
- Step by step assembly guide
This is to ensure that remote partner labs and other public shareholders are able to implement and adapt the resulting project.
12. How will your project address GOSH’s values of diversity and inclusion?
This project was created to address the lack of frugal yet full-featured options in the colony picking space. Few dedicated colony picking instruments currently exist. Those few that exist are costly, priced above 20000 USD even on the resale market.¹
The lack of these options restricts the possibilities of labs in lower to medium income countries with regards to high throughput colony picking. In addition, turn around times for equipment maintenance and repair are often issues with equipment that are imported with technical support usually based in overseas countries. Having a frugal, locally built picker solves this problem since this equipment can be maintained and repaired by local or even in-house repair crews.
This equipment would also benefit community labs where person-hours and resources are limited. A colony picker can handle multiple plates in a session, saving time for the users who have to share space and equipment. Having these machines in the labs also allows greater freedom for remote teams in other locations, allowing them to perform high throughput colony picking, screening and downstream assays alongside their other research tasks. Because they enable randomly arranged colonies to be programmatically and traceably ordered into a format amenable to high-throughput and automated processing, colony pickers also fit well into the workflow of an integrated frugal biofoundry of the type Friendzymes aims to build and validate, which will enable low-cost, high-capacity biological design-build-test cycles and strain engineering. Such frugal biofoundries will greatly lower the barriers to developing biotechnological productive capacity for resource-constrained people, teams, communities and countries.
¹ Examples of preexisting commercial solutions include: RapidPick™ Series (Hudson Robotics Inc), Pix Series (Molecular Devices LLC), PetriPlater Series (Tecan AG), Pickolo™ addon for Tecan robots (SciRobotics Ltd.), ROTOR + PIXL (Singer Instruments).
13. Are there any conflicts of interest that you wish to declare?
None.
14. Describe your experimental plan, including any new technologies or tools to be developed.
For the first phase, an imaging module with a picker prototype is put together to image a plate. The imaging module will be based on a raspberry pi microcomputer and camera together with illumination modules (white light or blue light). We will leverage the OpenCFU software package to identify the colony positions in the plate image relative to a coordinate system, and the OT2_MoClo_JoVE GitHub repo to generate colony coordinates that can be translated to a colony picking protocol on the Opentrons OT-2. This module will be evaluated and tested according to its capability of automatic colony location identification in the captured images. Once this setup is fully tested and shown to be viable, the imaging module design is finalized as a standalone module and a prototype gantry test rig is then developed to evaluate and correct the accuracy and precision of the resultant coordinates.
Colony picking with a pipetting robot like the OT-2 is sub-optimal, because of the relatively high cost and plastic waste generated by picking each individual colony with a different pipette tip. Thus, while building plate imagers, imaging plates and demonstrating/validating colony picking with the OpenTrons OT-2, we will simultaneously continue our work to design a dedicated open source colony picking robot, based on the OpenWorkStation framework, that uses either sterilizable, plastic filament that is cut between colony picks, or a thin extendable metal rod that is washed and sterilized between colony picks. By the end of the first phase, we will have design plans for prototyping this instrument.
For the second phase, we will build and test prototypes of the dedicated colony picking robot. We will aim to adapt and modify the FluoPi into an OpenWorkstation module that can interface directly with the colony picking module and with a plate transfer module for moving plates directly from the imager to the colony picker, creating a hands-free, single-set-up system that images, picks colonies and transfers them to specified output plate formats.The picking machine will be evaluated and tested according to its capability to pick the proper coordinates informed by the imaging module, which will involve proper calibration protocols. Finally, the machine will be tested with different kinds of colonies and setups.
15. How will the work you describe be performed within the budget and time period allocated for the initial Phase I award? This should include project work time, ramp up and required reporting.
This project is amenable to splitting into discrete sub-components that can be worked on in the different locations and then assembled for final testing. Most of the structural fabrication assembly will be done in the primary locations with concurrent integration effort being done by partner labs. When given the go-ahead, the contributors will decide on a timeline based on GOSH regulations and the contributors’ own capacity. After that, progress reports are to be done every week with progress to be documented in the Github repository. Supply and production issues are also documented using Github’s issue tracker.
Work shall be mainly split between the Boston, Chicago, British Columbia, and the Philippine working sites. The Boston and Philippine locations will be working on the initial hardware design and assembly. The Chicago and BC sites will then validate the colony identification workflow based on the imaging module. Once the workflow is validated, the Boston and Philippine sites shall benchmark the colony identification workflow on the assembled hardware, with emphasis taken on the precision and accuracy of the picker in relation to the generated coordinates. The hardware is then modified accordingly to bring the assembly up to an acceptable precision.
16. What essential milestones will you generate during your Phase I award?
By the end of the first funding phase, (1) the imaging module will be able to determine the viability of a plate for colony picking and the local coordinates of the isolated colonies in cartesian coordinates relative to a specified corner/edge location of the plate the colonies are on; and (2) OpenTrons OT-2 protocols will be demonstrated that translate these coordinates into a program to pick the specified colonies and transfer them to a specified set of output plates, generating plate maps recording identities and plate well / coordinate locations for each cloned colony on each output plate. Finally, (3) the design plan for prototyping the dedicated colony picking instrument will be finalized, including construction of at least one proof of concept of the sterilizer module and an OpenWorkStation gantry for later integration into the larger structure.
17. If Phase I is successfully completed, what are the next steps?
Upon successful completion of Phase I, the development team will proceed with final design of, followed by complete build-out of the assembly line system, including picking and sterilizing modules, transport module, cartesian-style gantry enclosure, and all other structural and electromechanical components. For budgeting breakdown after Phase I’s completion, please see 18.
18. Please include a brief breakdown of allowable direct costs under the following categories: personnel, supplies, subcontracts, travel, and other expenses.
Here are the allowable direct costs for this project with expanded details below. No travel or subcontracts are envisioned.
| Cost | Estimated Cost |
|---|---|
| Personnel | 915.00 |
| Supplies | 3,500.00 |
| Total | 4,515.00 |
| *see expanded table for additional details |
Personnel Estimates
| Module | Stage | Associated Activities | Estimated Labor Hours (blocking) | Estimated Labor Costs |
|---|---|---|---|---|
| Phase 1 | ||||
| Hardware - Structural | Design | Fitting of image module to openWorkstation footprint, Generation of final extrusion cut lengths, compilation of documentation | 9 | 135.00 |
| Hardware - Structural | Build | Printing/Cutting of structural Components, Assembly of Unit | 2 | 30.00 |
| Hardware - Structural | Test | Mechanical and load testing of assembly | 5 | 75.00 |
| Hardware - Electronics | Design | Generation of necessary gerber files, PCB rerouting(as needed) | 10 | 150.00 |
| Hardware - Electronics | Build | Assembly of parts, Hand soldering or small scale manufacture (i.e JLPCB) as needed. | 1.5 | 22.5 |
| Hardware - Electronics | Test | Continuity and PCB checks, Tolerance checks | 7.5 | 112.5 |
| Software | Design | Prototyping of PAML Specialization, Prototyping kinematics translation layer, GCODE compliance tests | 9 | 135.00 |
| Software | Build | Configuration, Build and Compile time | 1 | 15.00 |
| Software | Test | GCODE compliance checks, Script generation checks, Kinematics tests | 10 | 150.00 |
| Subtotal | 55 | 825.00 | ||
| Allowable Time Overruns | 6 | 90.00 | ||
| Total | 61 | 915.00 |
Supply Estimates
| Item | Estimated Cost |
|---|---|
| Structural Fasteners and Hardware | 120.00 |
| 3D Printed Parts | 300.00 |
| Acrylic Plates | 830.00 |
| RPi and associated attachments | 700.00 |
| Discrete Electronics/ PCB manufacturing and Components | 450.00 |
| Prototype - Gantry System | 500.00 |
| Prototype - Sterilizer | 200.00 |
| Prototype - Picker head | 300.00 |
| Miscellaneous | 200.00 |
| Total | 3,500.00 |