Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.
Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.
复制标题
DOI：
10.1002/cbic.202100507
复制
发表时间：
2022-04-05
期刊：
影响因子：
3.2
通讯作者：
Billerbeck, Sonja
中科院分区：
文献类型：
--
作者：
Kuiper, Bastiaan P.;Prins, Rianne C.;Billerbeck, Sonja
定制文库的构建对于合理的蛋白质工程和定向进化至关重要。由数千个用户定义的序列（长度高达350个碱基）组成的阵列合成寡核苷酸池已成为低成本的商业可用DNA来源。 这些池的成本≤ 10%（取决于错误率和长度）的其他商业来源的定制DNA，这种显着的成本差异可以决定是否可以实现酶工程项目在给定的研究预算。然而，虽然便宜，但寡核苷酸池确实遭受单个寡核苷酸的低浓度和相对高的错误率。已经开发了几种专门利用寡核苷酸池的强大技术，并证明其对下一代蛋白质和途径工程策略有价值甚至是必不可少的，例如序列功能映射，酶最小化或从头设计。在这里，我们巩固了这些技术及其应用的知识，以促进蛋白质工程社区内的寡聚池的使用。 构建大型变异库对于合理的蛋白质工程、定向进化和蛋白质从头设计至关重要，但受到DNA合成相对高成本的限制。在这里，我们整合了允许使用阵列合成的寡核苷酸池作为DNA输入的方法-一种廉价但容易出错的DNA来源。
The construction of custom libraries is critical for rational protein engineering and directed evolution. Array‐synthesized oligo pools of thousands of user‐defined sequences (up to ∼350 bases in length) have emerged as a low‐cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next‐generation protein and pathway engineering strategies, such as sequence‐function mapping, enzyme minimization, or de‐novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community. The construction of large variant libraries is critical for rational protein engineering, directed evolution, and protein de novo design, but is constrained by the relatively high cost of DNA synthesis. Here, we consolidate methods that allow using array‐synthesized oligo pools as DNA input – a cheap but error‐prone source of DNA.
登录
查看更多内容
影响因子：
14.9
作者：
Gibson DG
通讯作者：
Gibson DG
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
48
作者：
Fowler, Douglas M.;Fields, Stanley
通讯作者：
Fields, Stanley
影响因子：
48
作者：
Hiatt, Joseph B.;Patwardhan, Rupali P.;Turner, Emily H.;Lee, Choli;Shendure, Jay
通讯作者：
Shendure, Jay
影响因子：
12.6
作者：
Ghindilis, Andrey L.;Smith, Maria W.;McShea, Andy
通讯作者：
McShea, Andy