寡核苷酸
顺序装配
金门
计算生物学
生物
DNA
转化(遗传学)
DNA微阵列
DNA连接酶
基因
遗传学
计算机科学
工程类
土木工程
跨度(工程)
基因表达
转录组
作者
Sean Lund,Vladimir Potapov,Sean R. Johnson,Jackson Buss,Nathan A. Tanner
标识
DOI:10.1021/acssynbio.3c00694
摘要
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 (DAD) 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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