Directed Evolution: Methods and Protocols (Methods in Molecular Biology, 2461)

✍ Scribed by Andrew Currin (editor), Neil Swainston (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 280
• Edition: 1st ed. 2022
• Category: Library

✦ Synopsis

This volume explores the latest techniques used by researchers to study directed evolution (DE) at each stage of the Design-Build-Test-Learn cycle. Chapters in this book cover topics such as designing overlap extension PCR primers for protein mutagenesis; antha-guided automation of Darwin assembly for the construction of bespoke gene libraries; rapid cloning of random mutagenesis libraries using PTO-Quickstep; and DE of glycosyltransferases by a single-cell screening method. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Cutting-edge and comprehensive, Directed Evolution: Methods and Protocols is a valuable resource for scientists and researchers who are interested in learning more about this field and incorporating these studies into new experimental workflows.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Designing Overlap Extension PCR Primers for Protein Mutagenesis: A Programmatic Approach
1 Introduction
2 Materials
2.1 Running Environment
2.2 Input Files
2.3 Algorithm
3 Methods
4 Notes
References
Chapter 2: Recombination of Single Beneficial Substitutions Obtained from Protein Engineering by Computer-Assisted Recombinati...
1 Introduction
2 Material
2.1 The Initial Enzyme Structure and Substitution List
2.2 Setup of the FoldX Plugin for YASARA
3 Methods
3.1 Load the PDB File of Wild Type Enzyme (Step 1)
3.2 Select the Target Substitution for Calculation of the Relative Folding Free Energy (ΔΔGfold, Step 2)
3.3 Set Parameters for FoldX (Step 3)
3.4 Collection of ΔΔGfold Results and Alignment with the CompassR Rule (Step 4)
3.5 Create a Recombination Plan for Beneficial Substitutions (Step 5)
4 Notes
References
Chapter 3: Nondegenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization
1 Introduction
1.1 Saturation Mutagenesis
1.2 Trinucleotide Phosphoramidites (TRIM)
1.3 MAX Randomization
1.4 ProxiMAX Randomization
1.5 Other Routes to Nondegenerate Saturated DNA Libraries
1.6 Library Size, Sequence Space, and Screening
2 Materials
3 Methods
3.1 MAX Randomization
3.2 Joining Multiple MAX Randomization Cassettes by Overlap PCR
3.3 Joining Multiple MAX Randomization Cassettes by Golden Gate Assembly
3.4 Denaturation Gradient PCR: A Useful Tip for MAX Randomization of Repetitive Sequences
3.5 ProxiMAX Randomization
3.6 NGS Data Analysis of Saturated Libraries: Part 1, Creating an Excel Data File
3.7 Analysis of Excel Data by Delimiting
3.8 Analysis of Excel Data Using the COUNTIF´´ Function 3.9 Analysis of Excel Data Using Find and Replace,LEFT,´´ and ``MID´´ Functions
4 Notes
References
Chapter 4: Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries
1 Introduction
2 Materials
2.1 Master Mixes and Reaction Buffer for Gilson Pipetmax (See Note 1)
2.2 Oligonucleotides (Table 1)
2.3 Streptavidin Cleanup
2.4 Type IIS Restriction Cloning and Library Transformation (See Note 1)
2.5 Additional Reagents and Solutions
2.6 Equipment
3 Methods
3.1 Library Template Preparation
3.2 Library Design
3.3 Library Construction with Antha
3.4 Library Capture (for Libraries Assembled with Biotinylated Primers)
3.5 Library Recovery (See Note 4)
3.6 Library Transformation
3.7 Barcoding Libraries: Rationale
3.8 Barcoding Libraries: Introducing Barcodes to Libraries
3.9 Assembling a Barcoded NGS Sample
4 Notes
References
Chapter 5: SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Variant Libraries f...
1 Introduction
1.1 Process Overview
2 Materials
3 Methods
3.1 Design
3.1.1 Design of DNA Oligonucleotides
3.1.2 Generation of Worklists
3.2 Build: Application of Worklists
3.2.1 WT Dilution
3.2.2 Mutant Oligo Pooling
3.2.3 Template Pooling
3.2.4 PCR1
3.2.5 WT Block Pooling
3.2.6 Error Correction of WT Blocks
Stepwise Hybridization
3.2.7 Error Correction
PCR Amplification of WT Error Corrected Blocks
3.2.8 Pooling of Mutated Blocks
3.2.9 Assembly of the Full-Length Gene(s): PCR2
3.3 Test: Sequencing Analysis
3.3.1 Sample Preparation
3.3.2 Analysis
4 Notes
References
Chapter 6: Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis
1 Introduction
2 Materials
2.1 Strains
2.2 Reagents, Media, and Plates
2.3 Enzymes
2.4 Equipment and Materials
3 Methods
3.1 Preparation of Parental DNA Plasmid(s)
3.2 Design of Custom and Mutagenic Oligonucleotides
3.3 Preparation of Combinatorial Mutagenic Libraries by Nicking Mutagenesis
3.4 Sequence Verification of Combinatorial Library
4 Notes
References
Chapter 7: GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Buffers
3 Methods
3.1 Mutagenic Primer Design
3.2 Amplification of Mutagenic Megaprimers Via Asymmetric PCR
3.3 Amplification of a Full-Length Variant Library Using the Mutagenic Megaprimers
4 Notes
References
Chapter 8: Rapid Cloning of Random Mutagenesis Libraries Using PTO-QuickStep
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Nucleic Acid
2.3 Error-Prone PCR
2.4 Iodine Cleavage
2.5 Megaprimer PCR
2.6 DpnI Digestion and DNA Purification
2.7 Additional Molecular Biology Components
2.8 Transformation and Clone Analysis
3 Methods
3.1 Primer Design
3.2 Amplification of DNA Fragment of Interest
3.3 Iodine Cleavage and DNA Purification
3.4 Megaprimer PCR
3.5 Transformation
3.6 Colony Analysis
4 Notes
References
Chapter 9: Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs
1 Introduction
2 Materials
2.1 Media and Solutions
2.2 Primers, Enzymes and Kits
2.3 Instruments
2.4 Strains and Plasmids
3 Methods
3.1 Promoter Design
3.2 Primers Design
3.3 Construction of Vectors for Expression of Reporter Proteins or Customized Proteins
3.4 Promoter Library Construction and Screening
4 Notes
References
Chapter 10: Application of Restriction Free (RF) Cloning in Circular Permutation
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Vectors
2.3 Circular Permutation Reaction and Analysis
2.3.1 Circular Permutation Cloning Reaction
2.3.2 Analysis
3 Methods
3.1 Circular Permutation Using RF Cloning
3.1.1 Circularization of Gene of Interest
3.1.2 RF Cloning Approach to Construct the Desired Circular Permutants
3.2 Analysis of Clones
4 Notes
References
Chapter 11: Site-Directed Mutagenesis Method Mediated by Cas9
1 Introduction
2 Materials
3 Methods
3.1 Expression and Purification of spCas9 (THIS CAN BE REPLACED with Commercial spCas9 Enzyme)
3.2 Preparation of DNA Templates for In Vitro Transcription of sgRNA
3.3 Transcription of sgRNA In Vitro
3.4 In Vitro Cas9-Mediated Digestion of Target Plasmid
3.5 T5 Exonuclease Mediated Assembly
4 Notes
References
Chapter 12: Directed Evolution of Transcription Factor-Based Biosensors for Altered Effector Specificity
1 Introduction
1.1 Mutagenesis Strategy for aTF Directed Evolution
1.2 Screening and Selection of Variant Libraries for aTF Directed Evolution
1.3 PCA Biosensor Design and Function
2 Materials
2.1 Library Transformation into E. coli Expression Strain
3 Methods
3.1 Library Transformation into E. coli Expression Strain
3.1.1 Preparation of Electrocompetent Reporter Expression E. coli Cells
3.1.2 Transformation of the Libraries by Electroporation
3.2 aTF Variant Library Counterselection by Fluorescence Activated Cell Sorting (FACS)
3.3 Hits Test and Validation
4 Notes
References
Chapter 13: A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzy...
1 Introduction
2 Materials
2.1 Bioconversion
2.2 MP-CE Separation
3 Methods
3.1 Conversion by P450 BM3
3.2 MP-CE Measurement
4 Notes
References
Chapter 14: Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method
1 Introduction
2 Materials
2.1 Biological and Chemical Materials
2.2 Equipment
3 Methods
3.1 Generation of Random Mutagenesis Library
3.2 Screening via Flow Cytometry
3.2.1 Cell Growth and FutA Expression
3.2.2 Cytometric Analysis and Cell Sorting
3.3 Recovery of Mutants in Enriched Libraries
3.4 Secondary Screening and Analysis
4 Notes
References
Chapter 15: Learning Strategies in Protein Directed Evolution
1 Introduction
2 Materials
2.1 Hardware
2.2 Algorithm, Software, or Platform
3 Methods
3.1 Learning Strategies in Directed Evolution
3.1.1 Experimental and Computational Approaches
3.1.2 Lessons Prior to Directed Evolution
3.1.3 Lessons During Directed Evolution
3.1.4 Lessons at the End of Directed Evolution
3.2 Epistasis in Directed Evolution
3.3 Machine Learning and Directed Evolution
3.3.1 Introduction
3.3.2 General Machine Learning Workflow
Models of Data
3.3.3 Datasets for a Machine Learning Project in Directed Evolution
Data Readiness Levels
Training, Validation, and Test Set
How Much Data Is Needed?
What Type Data Is Needed?
3.3.4 Feature Extraction from Sequences and Structures
3.3.5 Machine Learning Approaches
What Type of ML Approach to Choose in Protein Directed Evolution?
Generalization, Overfitting, and Underfitting
3.3.6 Performance Metrics
References
Index