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Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems that perform the desired functions. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process has received serious attention in recent years. Importantly, automating DNA assembly enables larger builds using less researcher time, increasing the accessible design space.However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT).For this purpose, we developed an open-source software package dnabot (https://github.com/BASIC-DNA-ASSEMBLY/dnabot). We demonstrate the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, exploring the promoter, ribosome binding site (RBS) and gene order design space for a 3-gene operon. All 88 constructs were assembled with high accuracy, at a cost of $1.50 -$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT making it accessible for most labs and democratising automated DNA assembly.

Abstract Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site (RBS) and gene order design space for a 3-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50 - $5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratising automated DNA assembly.

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.

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