High-throughput, cost-effective verification of structural DNA assembly

Abstract

DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.

Engineering biological systems using automated biofoundries

“…Recently, liquid handlers coupled with high-throughput capillary electrophoresis has resulted in the implementation of a high-throughput genotype verification process (Dharmadi et al, 2014). For whole-genome (re-) sequencing and transcriptomic analysis, next-generation sequencing (NGS) has become routine practices.…”

“…Amyris is one of the pioneers in streamlining the DBTL cycles for engineering industrial microbes. A few technologies have been developed to improve the robustness and lower the cost of DNA assembly (de Kok et al, 2014) and construct verification (Dharmadi et al, 2014; Shapland et al, 2015). In the same space, startup companies such as Ginkgo Bioworks and Zymergen are also growing rapidly.…”

Developing Commercial Production of Semi-Synthetic Artemisinin, and of β-Farnesene, an Isoprenoid Produced by Fermentation of Brazilian Sugar

“…recent advances in the application of design automation, i.e., the use of software, hardware and robotics 22 have enabled the creation and screening of hundreds of thousands of strain variants (created by both design and random mutagenesis) for the properties required for commercial production of β-farnesene. Notable enabling technologies developed for routine usage include rapid and reliable assembly of large (i.e., multiple kilobase) deoxyribonucleic acid (DNA) constructs; [23][24][25] high throughput, cost effective, verification of structural DNA assemblies by both initial restriction digest 26 and by low-cost DNA sequencing; 27 and whole genome sequencing of yeast strains. 28 In addition, there is a need to effectively identify the best new strains (akin to panning for gold!)…”

Recent advances in DNA assembly technologies

“…Application of microfluidics in molecular biology has made promising advances which may turn out to be the enabling technologies for high-throughput DNA assembly confirmation (Mueller , et al , 2000, Dorfman , et al , 2013). Taking advantage of high throughput capillary electrophoresis, a cost-effective method for verification of DNA constructs has been reported (Dharmadi , et al , 2014). With the decreasing cost of DNA sequencing (Shendure & Ji, 2008, Clarke , et al , 2009), it may also become practical to directly sequence all assembled DNA molecules in the future.…”