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bioRxiv · 10.1101/2023.11.20.567888

Highly parallelized construction of DNA from low-cost oligonucleotide mixtures using Data-optimized Assembly Design and Golden Gate

Lund, S.·Potapov, V.·Johnson, S. R.·Buss, J.·Tanner, N. A.
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Abstract

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here we apply Data-optimized Assembly Design to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: 1) Amplification of parts belonging to individual assemblies in parallel from a single pool; 2) Golden Gate Assembly of parts for each construct; and 3) Transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods which require stability of extensive single-stranded DNA overhangs.

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Lund, S., Potapov, V., Johnson, S. R., Buss, J., Tanner, N. A.. 2023-11-20. Highly parallelized construction of DNA from low-cost oligonucleotide mixtures using Data-optimized Assembly Design and Golden Gate. https://doi.org/10.1101/2023.11.20.567888

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