Slowpoke: An Automated Golden Gate Cloning Workflow for Opentrons OT-2 and Flex

In synthetic biology, DNA assembly is a routine process where increasing demands for standardization, high-throughput capacity, and error-free execution are driving the development of accessible, automated solutions. Here, we present Slowpoke, a user-friendly and flexible workflow for Golden Gate-based cloning designed for the popular entry-cost, open-source liquid-handling platforms Opentrons OT-2 and Flex. Slowpoke automates the key steps of the DNA assembly process, including cloning, <i>Escherichia coli</i> transformation, plating, and colony PCR, requiring user intervention primarily for colony picking and plate transfers. To further simplify the usage, we developed a free graphical user interface (GUI), available at https://slowpoke.streamlit.app/, which enables rapid protocol generation through simple file uploads. We validated the workflow using two Golden Gate-based toolkits, the MoClo Yeast Toolkit (YTK), and SubtiToolKit (STK). High assembly efficiencies were achieved across platforms for basic transcript unit constructions: 17/17 positive colonies with YTK on OT-2, 11/12 on Flex, and 8/13 with STK on OT-2. High-throughput assemblies were also performed with six parts in Flex using YTK-compatible parts, and 55 out of 57 combinations resulted in correct constructs. These results confirm the robustness and adaptability of the workflow across toolkit complexity and automation platforms. The Slowpoke suite, including code scripts and templates, is freely available at https://github.com/Tom-Ellis-Lab/Slowpoke, offering an accessible and modular solution for automating Golden Gate cloning in synthetic biology laboratories.