Team:UPenn/Implementation
IMPLEMENTATION PLAN
The steps we took to implement the OptoReader:
1. Research limitations to current optogenetic research and opto-tools to work with
In discussion with Dr. Jose Avalos, Dr. Brian Chow, and Dr. Lukasz Bugaj along with our graduate student mentors Will, David, Gabby, and Saurabh, we discussed current research methods for optogenetics. We identified that there were no high-throughput, dynamic, and cost-effective methods of simultaneous reading, writing, and feedback control for optogenetic experiments.
2. Train team members in required software and hardware
In order to ideate a solution, we recognized that we needed to be versed and trained in software and hardware skills. We went through a mini bootcamp in Python, Arduino/C++, Raspberry Pi, and KiCad for the first 3 weeks of our project.
3. Design preliminary product hardware
We designed a preliminary version of a PlateReader that would couple with the optoPlate-96 to optically stimulate cells, read cellular output, and adjust stimulation patterns based on the live biological state in a 96-well format.
4. Create software and Graphical User Interface mock-up
In order for researcher’s to use the OptoReader, it needs to be controlled by a software package that includes Arduino scripts and Python code that controls the detailed protocols with precision and accuracy. In order to make our device accessible to a range of researchers, we needed to create a Graphical User Interface to easily put in protocol specifications without any prior knowledge of software.
5. Build first iteration of device and test with software
We ordered the designed Printed Circuit Boards and soldered on the components to build the first iteration of our device. This allowed us to also test our preliminary software architecture and bring to light features that did not work and that we wanted to add.
6. Create subsequent device iterations and adjust code in response to user feedback
We went through the Design, Built, Test, and Learn engineering cycle multiple times to create a version of the device to implement in biological systems. See our Engineering page for a detailed description of some of our cycles.
7. Test device detection limits in preliminary bacteria
Once we had a functioning device, the next step was to test its limits of detection for fluorescence readings and optical density readings. See our Experiments page for visualization of our experiment data.
8. Calibrate device using biological experiments and modeling
We then calibrated the different components of the device on both plates so that researchers can compare findings in different wells within the plate and trust that cell stimulation and output reading is consistent. See our Experiments page for visualization of our experiment data.
9. Test device robustness in a shaking bacterial incubator
In order for researchers to fully implement our device, it must function in conditions suitable for bacterial growth, i.e. at 37C with constant shaking, a typical condition used for bacterial growth. We tested the OptoReader in these conditions for overnight experiments to assess its robustness.
10. Test reading and writing capabilities of the OptoReader using optogenetic transcription.
We tested the ability of the OptoReader to stimulate optogenetic transcription and simultaneously measure resultant fluorescence and OD. We used the previously described pDawn and pDusk optogenetic transcription modules to control transcription (Lawani et. al, 2021). See our Experiments page for visualization of our experiment data.
11. Implement feedback mechanisms
Our device can uniquely allow closed=loop feedback control of optogenetic experiments. This means the device can detect the current biological state and adjust the stimulation pattern according to a function preset by the user. We tested feedback with the pDawn/pDusk system. See our Experiments page for visualization of our experiment data.
12. Properly document all hardware, software, and user design aspects
To ensure that our open-source device is broadly available, we put our software package on GitHub and document the printed circuit boards in the Hardware section.
13. Test in user-cases at other research laboratories
We are in the process of giving our device to other laboratories to test themselves in their own labs and with unique biological systems. We anticipate meaningful results in the form of potential improvements to our User Interface and device capabilities.
References
1. Lalwani, M. A., Kawabe, H., Mays, R. L., Hoffman, S. M., & Avalos, J. L., Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synthetic Biology, 2021. 10(8), 2015-2029.
