基于质粒的一锅法饱和诱变
Plasmid-based one-pot saturation mutagenesis.
作者信息
Wrenbeck Emily E, Klesmith Justin R, Stapleton James A, Adeniran Adebola, Tyo Keith E J, Whitehead Timothy A
机构信息
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, USA.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
出版信息
Nat Methods. 2016 Nov;13(11):928-930. doi: 10.1038/nmeth.4029. Epub 2016 Oct 10.
Abstract
Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.
摘要
深度突变扫描是一种用于研究大量突变体功能后果的基础工具，但需要一种更高效、更易操作的方法来构建用户定义的诱变文库。在此，我们介绍切口诱变，这是一种在常规制备的质粒双链DNA上进行的强大、单日、一锅法饱和诱变方法。该方法可用于产生全面的或单位点或多位点饱和诱变文库。
相似文献
1
Plasmid-based one-pot saturation mutagenesis.基于质粒的一锅法饱和诱变
Nat Methods. 2016 Nov;13(11):928-930. doi: 10.1038/nmeth.4029. Epub 2016 Oct 10.
2
User-defined single pot mutagenesis using unamplified oligo pools.用户定义的单个小瓶诱变使用未扩增的寡聚池。
Protein Eng Des Sel. 2019 Sep 10;32(1):41-45. doi: 10.1093/protein/gzz013.
3
Uracil-USE, an improved method for site-directed mutagenesis on double-stranded plasmid DNA.尿嘧啶使用法（Uracil-USE），一种用于双链质粒DNA定点诱变的改进方法。
Biotechniques. 1995 Mar;18(3):370-2.
4
[Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].[利用设计的限制性内切酶辅助诱变对全长质粒DNA进行快速定点诱变]
Sheng Wu Gong Cheng Xue Bao. 2009 Feb;25(2):306-12.
5
Parallel assembly for multiple site-directed mutagenesis of plasmids.质粒多位点定向突变的并行组装。
Anal Biochem. 2012 Nov 1;430(1):65-7. doi: 10.1016/j.ab.2012.07.029. Epub 2012 Aug 8.
6
Polishing the craft of genetic diversity creation in directed evolution.定向进化中遗传多样性创造技术的优化。
Biotechnol Adv. 2013 Dec;31(8):1707-21. doi: 10.1016/j.biotechadv.2013.08.021. Epub 2013 Sep 6.
7
Patch cloning method for multiple site-directed and saturation mutagenesis.多位点定点突变和饱和突变的补丁克隆方法。
BMC Biotechnol. 2013 Oct 29;13:91. doi: 10.1186/1472-6750-13-91.
8
PFunkel: efficient, expansive, user-defined mutagenesis.PFunkel：高效、广泛、用户定义的诱变。
PLoS One. 2012;7(12):e52031. doi: 10.1371/journal.pone.0052031. Epub 2012 Dec 17.
9
Site-Directed Mutagenesis Method Mediated by Cas9.基于 Cas9 的定点突变方法。
Methods Mol Biol. 2022;2461:165-174. doi: 10.1007/978-1-0716-2152-3_11.
10
The amidase regulatory gene (amiR) of Pseudomonas aeruginosa.铜绿假单胞菌的酰胺酶调节基因（amiR）。
J Gen Microbiol. 1987 Aug;133(8):2041-52. doi: 10.1099/00221287-133-8-2041.
引用本文的文献
1
CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.CRISPR/Cas系统引导的无序列限制的质粒诱变
Fundam Res. 2022 Jul 15;5(4):1481-1487. doi: 10.1016/j.fmre.2022.06.017. eCollection 2025 Jul.
2
SNAP-tag2 for faster and brighter protein labeling.用于更快速、更明亮蛋白质标记的SNAP-tag2。
Nat Chem Biol. 2025 Jul 3. doi: 10.1038/s41589-025-01942-z.
3
Massively parallel genetic perturbation suggests the energetic structure of an amyloid-β transition state.大规模平行基因扰动揭示了淀粉样β蛋白过渡态的能量结构。
Sci Adv. 2025 Jun 13;11(24):eadv1422. doi: 10.1126/sciadv.adv1422. Epub 2025 Jun 11.
4
Computational design of dynamic biosensors for emerging synthetic opioids.用于新型合成阿片类药物的动态生物传感器的计算设计
bioRxiv. 2025 May 17:2025.05.15.654300. doi: 10.1101/2025.05.15.654300.
5
Unusually Broad-spectrum small-molecule sensing using a single protein scaffold.利用单一蛋白质支架实现异常广谱的小分子传感。
bioRxiv. 2025 May 15:2025.05.15.654352. doi: 10.1101/2025.05.15.654352.
6
Deep mutational scanning reveals a de novo disulfide bond and combinatorial mutations for engineering thermostable myoglobin.深度突变扫描揭示了用于工程化耐热肌红蛋白的一个新生二硫键和组合突变。
Protein Sci. 2025 May;34(5):e70112. doi: 10.1002/pro.70112.
7
Latent activity in TnpB revealed by mutational scanning.通过突变扫描揭示的TnpB潜在活性
bioRxiv. 2025 Feb 16:2025.02.11.637750. doi: 10.1101/2025.02.11.637750.
8
Epitope mapping via in vitro deep mutational scanning methods and its applications.通过体外深度突变扫描方法进行的表位作图及其应用
J Biol Chem. 2025 Jan;301(1):108072. doi: 10.1016/j.jbc.2024.108072. Epub 2024 Dec 14.
9
RecombiCraft library construction: A novel method for DNA library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture.重组工艺文库构建：一种使用非酶促单步DNA重组和液体培养进行DNA文库克隆与扩增的新方法。
PLoS One. 2024 Dec 2;19(12):e0312188. doi: 10.1371/journal.pone.0312188. eCollection 2024.
10
Beyond Single Clones: High-Throughput Sequencing in Antibody Discovery.超越单克隆：抗体发现中的高通量测序
Cold Spring Harb Protoc. 2025 Jan 2;2025(1):pdb.top107772. doi: 10.1101/pdb.top107772.
本文引用的文献
1
Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli.大肠杆菌中左旋葡聚糖利用途径的综合序列-通量图谱
ACS Synth Biol. 2015 Nov 20;4(11):1235-43. doi: 10.1021/acssynbio.5b00131. Epub 2015 Sep 22.
2
Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.利用全面诱变和深度测序进行快速精细构象表位图谱分析
J Biol Chem. 2015 Oct 30;290(44):26457-70. doi: 10.1074/jbc.M115.676635. Epub 2015 Aug 20.
3
High-resolution sequence-function mapping of full-length proteins.全长蛋白质的高分辨率序列-功能图谱
PLoS One. 2015 Mar 19;10(3):e0118193. doi: 10.1371/journal.pone.0118193. eCollection 2015.
4
Evolvability as a function of purifying selection in TEM-1 β-lactamase.TEM-1 β-内酰胺酶中纯化选择作用下的可进化性。
Cell. 2015 Feb 26;160(5):882-892. doi: 10.1016/j.cell.2015.01.035.
5
Massively parallel single-amino-acid mutagenesis.大规模平行单氨基酸诱变
Nat Methods. 2015 Mar;12(3):203-6, 4 p following 206. doi: 10.1038/nmeth.3223. Epub 2015 Jan 5.
6
The interrelationship between promoter strength, gene expression, and growth rate.启动子强度、基因表达和生长速率之间的相互关系。
PLoS One. 2014 Oct 6;9(10):e109105. doi: 10.1371/journal.pone.0109105. eCollection 2014.
7
Measuring the activity of protein variants on a large scale using deep mutational scanning.利用深度突变扫描大规模测量蛋白质变体的活性。
Nat Protoc. 2014 Sep;9(9):2267-84. doi: 10.1038/nprot.2014.153. Epub 2014 Aug 28.
8
Deep mutational scanning: a new style of protein science.深度突变扫描：一种新的蛋白质科学研究方法。
Nat Methods. 2014 Aug;11(8):801-7. doi: 10.1038/nmeth.3027.
9
Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes.体内激酶的全面突变扫描揭示了底物依赖性的适应性景观。
Nucleic Acids Res. 2014 Aug;42(14):e112. doi: 10.1093/nar/gku511. Epub 2014 Jun 9.
10
A comprehensive, high-resolution map of a gene's fitness landscape.一张关于某个基因适应度景观的全面、高分辨率图谱。
Mol Biol Evol. 2014 Jun;31(6):1581-92. doi: 10.1093/molbev/msu081. Epub 2014 Feb 23.
Zotero 插件