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

## TL;DR

Slowpoke is an automated workflow for Golden Gate cloning on Opentrons platforms, streamlining DNA assembly with a user-friendly interface and high success rates.

## Contribution

A flexible, automated Golden Gate cloning workflow with a GUI for Opentrons OT-2 and Flex, validated across toolkits and platforms.

## Key findings

- High assembly efficiencies were achieved with 17/17, 11/12, and 8/13 correct constructs using different toolkits and platforms.
- 55 out of 57 high-throughput assemblies with six parts on Flex resulted in correct constructs.
- The workflow is robust and adaptable across toolkit complexity and automation platforms.

## Abstract

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, Escherichia coli 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.