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经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序
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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Please note that all translations are automatically generated.
Click here for the English version.
经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序
DOI:
10.3791/54119-v
•
11:36 min
•
July 03, 2016
•
Michael Allen Stiffler
,
Subu K Subramanian
,
Victor H Salinas
,
Rama Ranganathan
1
Green Center for Systems Biology
,
University of Texas Southwestern Medical Center
Chapters
00:05
Title
00:43
Synthesis of NNS Sub-libraries for Each Amino Acid Position by Two-step PCR Site-directed Mutagenesis
01:53
Cloning of NNS Sub-libraries into Selection Vectors
03:47
Selection of the TEM-1 Whole-Protein Saturation Mutagenesis Library for Antibiotic Resistance
05:51
High Throughput Sequencing to Determine Fitness Effects of Mutations
08:44
Results: High Throughput Sequencing from Whole-Protein Saturation Mutagenesis
10:16
Conclusion
Summary
Automatic Translation
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Automatic Translation
我们提出了一个协议为利用高通量测序蛋白的综合单站点饱和突变库功能评估。重要的是,这种方法采用正交引物对复图书馆建设和测序。提供了使用在氨苄青霉素的临床相关剂量选择的TEM-1β内酰胺酶代表性结果。
Tags
Keywords: Saturation Mutagenesis
High-throughput Sequencing
Protein Engineering
Molecular Evolution
Biochemical Constraints
PCR
Library Construction
Multiplexing
NNS Sublibrary
Ligation
Electroporation
E. Coli Transformation
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