Automated workflow for genotyping individual transposon library variants.

Area of Science:

• Genome Engineering
• Synthetic Biology
• Laboratory Automation

Background:

• High-throughput screening is crucial for genome engineering and synthetic biology but often relies on expensive equipment.
• Genotyping individual clones in transposon libraries is labor-intensive, despite advances in population-level screening.
• Open-source liquid handlers offer accessibility but require programming expertise or validated protocols.

Purpose of the Study:

• To develop a step-by-step automated workflow for individual variant genotyping of transposon insertion libraries.
• To address challenges of cost, programming expertise, and protocol availability in laboratory automation.
• To utilize an open-source OT-2 liquid handler and a custom Python package for efficient and reproducible genotyping.

Main Methods:

• An automated protocol with six modular steps: colony picking, counter-selection, PCR assays, and sequencing with custom Python annotation.
• Utilized standardized vectors (SEVA guidelines) and Lab Automation Protocol (LAP) scripts for reproducibility.
• Demonstrated workflow on a transposon insertion library in Pseudomonas putida KT2440.

Main Results:

• Validated automated workflow for reliable genotyping of individual variants within 4-5 days.
• Achieved successful identification of genomic insertion sites for 40 out of 46 variants with no spurious integrations detected.
• Demonstrated the robustness, accessibility, and scalability of the open-source workflow.

Conclusions:

• The automated workflow reliably reduces hands-on time and human error in genotyping.
• Its modular and standardized design promotes protocol reuse, sharing, and broader access to open-source automation.
• Provides a scalable and reproducible foundation for variant genotyping in genome engineering and synthetic biology.