AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing platform using a compact benchtop sequencer

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genomics

Background:

• Plasmid sequence verification is crucial in synthetic biology.
• Next-generation sequencing (NGS) library preparation often utilizes Tn5 transposase.
• In-house recombinant Tn5 production can be labor-intensive.

Purpose of the Study:

• To develop a simplified, in-house Tn5-based plasmid sequencing workflow.
• To optimize the production of active Tn5 transposase.
• To evaluate a complete sequencing and data analysis pipeline for laboratory-scale applications.

Main Methods:

• Fusion of His10 tag and protein A for efficient Tn5 transposase purification.
• Exonuclease-based genomic DNA digestion of E. coli lysate for plasmid DNA.
• Data analysis using Illumina iSeq100 for de novo assembly, reference mapping, and annotation.

Main Results:

• A His10-Protein A fusion tag is sufficient for active Tn5 transposase production.
• Successful plasmid sequencing from E. coli lysate using the optimized workflow.
• Demonstrated efficacy of the data analysis pipeline for sequence assembly and annotation.

Conclusions:

• An easy-to-purify Tn5 transposase enables a streamlined plasmid sequencing platform.
• The proposed workflow simplifies laboratory-scale synthetic biology applications.
• Integration with compact benchtop sequencers like AistSeq enhances accessibility.