Assemblatron: An Automated Workflow for High-Throughput Assembly of Big-DNA Libraries

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genomics

Background:

• Synthesizing large DNA molecules (50 kb+) is crucial for genome engineering, metabolic engineering, and synthetic genomics.
• Current methods for assembling custom big DNAs are slow, expensive, and labor-intensive, hindering progress in the field.

Purpose of the Study:

• To present a set of improvements to yeast-based DNA assembly methods for medium-to high-throughput big DNA experiments.
• To reduce the cost, effort, and time required for assembling large DNA constructs.

Main Methods:

• Development of two novel vector systems: Jack In the Box (JIB) and Selection of URS Recombinational Excision (SURE).
• Improvement of yeast transformation efficiency and development of an automation pipeline for segment mixing.
• Establishment of phenotype-driven assays for rapid reporting of assembly frequencies.
• Implementation of oligo-guided architecture in yeast (OLIGARCHY) for combinatorial assembly and segment reuse.

Main Results:

• JIB and SURE vector systems significantly reduced background colonies, minimizing screening efforts.
• High success rate in assembling diverse big DNA libraries using OLIGARCHY, with one person assembling 7 Mb of DNA in 7 days.
• Efficient conversion of oligonucleotides into gene-sized double-stranded DNA segments using the SURE vector, bypassing PCR amplification.

Conclusions:

• The presented improvements enable efficient, cost-effective, and high-throughput assembly of large DNA molecules in yeast.
• These advancements facilitate rapid construction of complex DNA libraries and variants for synthetic biology applications.
• The SURE vector offers an improved method for converting oligonucleotides into functional DNA segments.