Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications

Abstract

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality

“…S1 A ). We used our previously reported computational DNA design algorithms (31, 32) and generated a synthesis-optimized genome design termed C. eth-2.0 . Cumulatively, we introduced 10,172 base substitutions and removed 5,668 synthesis constraints (Table 2).…”

“…Furthermore, fundamental functions encoded within the essential genomes can be identified. In addition to the base substitutions introduced for synthesis streamlining, we used computational sequence design algorithms (31, 32) to deliberately add 123,141 base substitutions within protein-coding sequences to yield the rewritten C. eth-2.0 design (Table 2) (33). In C. eth-2.0 , we replaced 56.1% of all codons by synonymous versions.…”

“…We computationally devised a four-tier DNA assembly strategy starting from 3- to 4-kb assembly blocks to build the complete C. eth-2.0 chromosome in yeast (32) (Fig. 2 A ).…”

“…On the level of de novo DNA synthesis, we herein demonstrate how chemical synthesis rewriting facilitates the genome synthesis process. To simplify the entire genome build process, we used sequence design algorithms (31, 32) and collectively introduce 10,172 base substitutions to remove 5,668 DNA synthesis constraints, including 1,233 repeats, 93 homopolymeric stretches, and 4,342 regions of high GC content. Successful low-cost synthesis and subsequent higher-order assembly of C. eth-2.0 into the complete chromosome exemplify the utility of our approach to rapidly produce designer genomes.…”

“…The streamlined C. eth-2.0 design contains a low amount of both synthesis constraints and unnecessary genetic features. To enable the retrosynthetic assembly route, C. eth-2.0 was partitioned into 3- to 4-kb DNA blocks using the previously published Genome Partitioner algorithm (32).…”