One of the great parts about having an automated robot is being able to precisely mix, deposit, and run reactions without much intervention. This year, a greater emphasis will be placed on utilizing the Opentrons to accelerate your final projects.
For this week, we’d like for you to do the following
While your idea doesn’t need to be set in stone, we would like to see core details of what you would automate. This is due right before class and does not need to be tested on the Opentrons for now.
Following up on my concept from week2, the construct I plan to make will have a scaffold module and a target module. Ideally, the construct will self-form with the target decorating the scaffold. One such example maybe a system of the SpyTag and SpyCatcher.
In making this construct, we will be able to test various parameters in the structure including: (a) size of scaffold building unit (b) linker size binding antibody. Trying various size of both parameters may be needed to get to a working structure.
A way to test an intact structure could be based on imaging. By combining fluorescent protein expression (say GFP & RFP ) on both modules (scaffold and target), we will be able to test when a structure was formed and possible stochiometry of the target module. Using lab automation can be useful in several parts once multiple DNA sequences for the optimization of bio-sponge are made:
Using automated liquid handler like Opentrons could be useful for such process in moving liquids for such large scale optimization. And Multi-well fluorescent microscopy with data analysis could be used to assess the performance.
General Overview: The paper “Luciferase Reporter Assay for NF-κB Activation Automated by an Open-Source Liquid Handling Platform” demonstrates the application of lab automation using the Opentrons platform to perform a luciferase assay for studying gene expression. The authors leverage the high sensitivity of luciferase to quantify NF-κB-driven transcriptional activity—a key factor in regulating genes involved in cell proliferation, differentiation, and survival. By integrating Opentrons into their workflow, they address the high reagent costs and limited throughput associated with traditional liquid handlers, making this method more accessible for diverse research settings.
In this work, the low-cost, open-source Opentrons robot automates multiple steps in a 96-well plate format, including HeLa cell culture preparation, transfection of an NF-κB luciferase reporter construct, treatment with varying concentrations of an NF-κB inducer (PMA), and the final measurement of luciferase activity using a plate reader. The automation streamlines the process and minimizes human error, which is critical when studying the nuanced effects of gene expression under different experimental conditions.
Findings: The study shows that using the Opentrons platform significantly reduces the time required for sample handling—with individual steps taking 5:19, 10:49, 20:54, and 27:08 minutes, respectively—compared to manual processing. Although some steps (such as initial cell preparation and plate transfers) remain manual, the overall workflow is markedly improved. The data revealed that increasing PMA concentrations led to a clear, reproducible increase in NF-κB-driven luciferase activity, as evidenced by a low coefficient of variation among replicates.
Relevant Figures:
Abstract Figure: This figure illustrates the overall workflow, highlighting the integration of the Opentrons robot in automating the assay steps.