Cell-Free Gene Expression: Methods and Protocols

Preface
	Part I: Tools for Cell-Free Expression Systems
	Part II: Applications of Cell-Free Expression Systems
Contents
Contributors
Part I: Tools for Cell-Free Expression Systems
	Chapter 1: Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production
		1 Introduction
		2 Materials
			2.1 Calibration
			2.2 Cell Growth
			2.3 DNA Extraction
			2.4 DNA Purification
			2.5 DNA Quantitation
			2.6 Cell-Free Protein Production
			2.7 Gel Electrophoresis
		3 Methods
			3.1 Instrument Calibration for Benchtop Incubators for Plate and Liquid Culture Growth
			3.2 Manufacturer´s Calibration for Fluorescence Plate Reader
			3.3 Calibration for Fluorescence Plate Reader for Automated DNA Quantitation
			3.4 Calibration for Fluorometer for Manual DNA Quantitation
			3.5 Calibration for Spectrophotometer for Manual Absorbance Measurements of DNA Using Spectrophotometry
			3.6 Calibration for Fluorescence Plate Reader for Cell-Free Protein Production
			3.7 Daily Maintenance of Automated Liquid Handler with Positive Pressure Module
			3.8 Liquid Class Development for Automated Methods Using a Gravimetric Approach
			3.9 Plate Culture
			3.10 Liquid Culture
			3.11 Pellet Cell Culture
			3.12 Manual DNA Extraction with Filter Columns: QIAprep Spin Miniprep Kit, or its Equivalent
			3.13 Automated DNA Extraction with Filter Plates: QIAprep 96 Turbo Miniprep Kit, or its Equivalent
			3.14 Manual DNA Extraction with Magnetic Beads: Mag-Bind Ultra Pure Plasmid DNA 96 Kit, or its Equivalent
			3.15 Automated DNA Extraction with Magnetic Beads: Mag-Bind Ultra Pure Plasmid DNA 96 Kit, or its Equivalent
			3.16 Manual DNA Purification with Filter Columns: PureLink PCR Purification Kit, or its Equivalent
			3.17 Automated DNA Purification with Filter Plates: PureLink Pro 96 PCR Purification Kit, or its Equivalent
			3.18 Manual DNA Quantitation with Fluorometry: Quant-iT Broad-Range dsDNA Assay Kit, or its Equivalent
			3.19 Automated DNA Quantitation with Fluorometry: Quant-iT Broad-Range dsDNA Assay Kit, or its Equivalent
			3.20 Manual DNA Qualification with Spectrophotometry
			3.21 Gel Electrophoresis of DNA Templates
			3.22 Cell-Free Protein Production: myTXTL Sigma 70 Master Mix, or its Equivalent
		4 Notes
		References
	Chapter 2: Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression
		1 Introduction
		2 Materials
			2.1 Agar Plate
			2.2 CFAI Workflow-Media Preparation
			2.3 2x YTPG Workflow-Media Preparation
			2.4 Cell Harvest
			2.5 CFPS Reaction
		3 Methods
			3.1 Agar Plate Inoculation
			3.2 CFAI Workflow-Inoculation and Cell Growth
			3.3 2x YTPG Workflow-Inoculation and Cell Growth
			3.4 Cell Harvest
			3.5 Extract Preparation
			3.6 CFPS Reaction
		4 Notes
		References
	Chapter 3: Preparation and Screening of Cell-Free Extract from Nongrowing Escherichia coli A19 Cells
		1 Introduction
		2 Materials
			2.1 Growth and Harvest of E. coli
			2.2 S30 Extract Preparation
			2.3 Cell-Free Protein Synthesis
		3 Methods
			3.1 Growth and Harvest of E. coli
			3.2 S30 Extract Preparation
			3.3 Cell-Free Protein Synthesis
		4 Notes
		References
	Chapter 4: Cell-Free Protein Synthesis Using Pichia pastoris
		1 Introduction
		2 Materials
			2.1 Preparation of Pichia pastoris Lysate
			2.2 Coupled In Vitro Transcription and Translation
			2.3 Determining Luciferase Production from Cell-Free Reactions
		3 Methods
			3.1 Preparation of Pichia pastoris Lysate
			3.2 Coupled In Vitro Transcription and Translation
			3.3 Determining Luciferase Production from Cell-Free Reactions
		4 Notes
		References
	Chapter 5: A Streptomyces-Based Cell-Free Protein Synthesis System for High-Level Protein Expression
		1 Introduction
		2 Materials
			2.1 Preparation of Streptomyces Cell Extracts
			2.2 Expression and Purification of Protein Translation Related Factors: IF1, IF2, IF3, EF-Tu, EF-Ts, EF-G, RF1, RF2, and  RRF
			2.3 Detection of Protein Yields
		3 Methods
			3.1 Cultivation of S. lividans
			3.2 Collection and Washing of Cells
			3.3 Disruption of Cells
			3.4 Construction of Plasmids for the Expression of Nine Translation Related Factors
			3.5 Expression and Purification of Nine Translation Related Factors
			3.6 Make Protein Standard Curve and Detect the Concentration of Nine Proteins
			3.7 Translation Related Factors Enhance EGFP Expression In Vitro
		4 Notes
		References
	Chapter 6: In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins
		1 Introduction
		2 Materials
			2.1 Cell-Free Expression
				2.1.1 HeLa Cell Culture
				2.1.2 HeLa Lysate Preparation
				2.1.3 Assembling TX-TL Coupled CFE Reaction
			2.2 Airfuge Fractionation Assay
			2.3 Membrane Protein Incorporation Assay
				2.3.1 SUPER Template Preparation
				2.3.2 Incorporation and Protease Protection Assay
			2.4 Vesicle Encapsulation System
		3 Methods
			3.1 Preparation of HeLa-Based CFE System
				3.1.1 HeLa Cell Culture
				3.1.2 HeLa Lysate Preparation
				3.1.3 Assembling Coupled Cell-Free Expression Reaction
			3.2 Airfuge Fractionation Assay
			3.3 Membrane Protein Incorporation Assay
				3.3.1 SUPER Template Preparation
				3.3.2 Membrane Protein Incorporation
				3.3.3 Protease Protection Assay
			3.4 Vesicle Encapsulation System
		4 Notes
		References
	Chapter 7: High-Throughput Experimentation Using Cell-Free Protein Synthesis Systems
		1 Introduction
		2 Materials
			2.1 Strain and Plasmid Construction for Whole-Cell Extract
			2.2 Whole-Cell Extract Preparation
			2.3 Preparation of Cell-Free Reaction Supplement
			2.4 Cell-Free Reaction Supplement Preparation
			2.5 Printing Platform Equipment and Consumables
		3 Methods
			3.1 Construction of BL-7S Strains
			3.2 Preparation of BL-7S Whole-Cell Extract
			3.3 Printing Platform Assembly
			3.4 Printer Head Fabrication
			3.5 Printing Calibration
				3.5.1 Calibration Theory
				3.5.2 Method for Individual Printer Head Calibration
		4 Notes
		References
	Chapter 8: Cell-Free Gene Expression from DNA Brushes
		1 Introduction
		2 Materials
			2.1 DNA Solutions
			2.2 Cleaning Slides with Base Piranha
			2.3 Coating Slides with a Photosensitive and Biocompatible Polymer Monolayer
			2.4 Biotin Surface-Patterns
			2.5 Preparation of the Reaction Chamber
			2.6 Assembly of DNA Brushes and Deposition of Antibodies
			2.7 Gene Expression Reaction
			2.8 Imaging
		3 Methods
			3.1 DNA Solutions
			3.2 Cleaning Slides with Base Piranha
			3.3 Coating Slides with a Photosensitive and Biocompatible Polymer Monolayer
			3.4 Biotin Surface-Patterns
			3.5 Preparation of the Reaction Chamber
			3.6 Assembly of DNA Brushes and Deposition of Antibodies
			3.7 Gene Expression Reaction
			3.8 Imaging
		4 Notes
		References
	Chapter 9: Efficient and Precise Protein Synthesis in a Cell-Free System Using a Set of In Vitro Transcribed tRNAs with Nucleo...
		1 Introduction
		2 Materials
			2.1 Affinity Purification of Proteins
			2.2 In Vitro Transcription of iVT tRNA
			2.3 Nucleotide Modification of iVT tRNA
			2.4 Protein Synthesis and Measurement of the Activity of Synthesized Product
		3 Methods
			3.1 Plasmid Designs
			3.2 Affinity Purification of Proteins
				3.2.1 Purification of TsaB and TrmD
				3.2.2 Purification of TsaC and TsaE
				3.2.3 Purification of MnmE and GlyA
				3.2.4 Purification of TsaD
				3.2.5 Purification of MnmC
				3.2.6 Purification of C5 Protein
			3.3 In Vitro Transcription of iVT tRNA
				3.3.1 Preparation of DNA Template
				3.3.2 In Vitro Transcription of iVT tRNA
			3.4 Nucleotide Modification of iVT tRNA
			3.5 Protein Synthesis with the PURE System
			3.6 Measurement of Synthesized DHFR Activity
		4 Notes
		References
	Chapter 10: Measurement of Transcription, Translation, and Other Enzymatic Processes During Cell-Free Expression Using PERSIA
		1 Introduction
		2 Materials
		3 Methods
			3.1 Basic PERSIA Method to Measure Real-Time Transcription/Translation from a Target Nucleic Acid
			3.2 PERSIA Method Measuring Real-Time Transcription/Translation and HIV Protease Enzymatic Activity in Parallel Reactions
		4 Notes
		References
Part II: Applications of Cell-Free Expression Systems
	Chapter 11: Cell-Free Noncanonical Redox Cofactor Systems
		1 Introduction
		2 Materials
			2.1 Purified Protein-Based Systems
				2.1.1 Purified Protein Preparation
				2.1.2 Purified Protein Reaction System
			2.2 Crude-Lysate Based Systems
				2.2.1 Crude-Lysate Preparation
				2.2.2 Determining the Molar Concentration of the Enriching Protein in the Crude Lysate
				2.2.3 Crude-Lysate Reaction System
		3 Methods
			3.1 Purified Protein-Based Systems
				3.1.1 Purified Protein Preparation
				3.1.2 Purified Protein-Based Biotransformation
			3.2 Crude Lysate-Based Systems
				3.2.1 Crude Lysate Generation
				3.2.2 Determining the Molar Concentration of the Enriching Protein in the Crude Lysate
				3.2.3 Crude Lysate Biotransformation
			3.3 System Adaptation and Design Considerations
		4 Notes
		References
	Chapter 12: Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping
		1 Introduction
		2 Materials
			2.1 Cell Extract Preparation
			2.2 Cell-Free Reactions
			2.3 Protein Quantification via Radioactivity
		3 Methods
			3.1 Cell Extract Preparation
				3.1.1 Cell Growth and Harvest
				3.1.2 Lysis and Clarification
			3.2 Cell-Free Protein Synthesis (CFPS)
			3.3 Protein Quantification
			3.4 Cell-Free Metabolic Engineering (CFME)
				3.4.1 CFME Assembly by  Hand
				3.4.2 Assembly Using an Echo 550 Automated Liquid-Handling Robot
		4 Notes
		References
	Chapter 13: Metabolomics Analysis of Cell-Free Expression Systems Using Gas Chromatography-Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Cell-Free Reaction Materials
			2.2 Protein Precipitation Materials
			2.3 GC-MS Data Acquisition
			2.4 Data Processing Software
		3 Methods
			3.1 CFE Reaction Assembly
			3.2 Protein Precipitation for Metabolomics Analysis
			3.3 Derivatization and Data Acquisition
			3.4 Data Processing
		4 Notes
		References
	Chapter 14: Liposome Preparation by 3D-Printed Microcapillary-Based Apparatus
		1 Introduction
		2 Materials
			2.1 3D-Printed Microfluidics Device
			2.2 Lipid-Oil Solution
			2.3 Biomimetic Lumen Chemistries
			2.4 Liposome Preparation Buffer
		3 Methods
			3.1 Preparation of Lipid-Oil Solution
			3.2 Assembly of 3D-Printable Microfluidics Device
			3.3 Liposome Preparation
			3.4 Example  Data
		4 Notes
		References
	Chapter 15: Microfluidic Production of Porous Polymer Cell-Mimics Capable of Gene Expression
		1 Introduction
		2 Materials
			2.1 Fabrication of SU-8 Patterned Silicon Wafers
			2.2 Production of Microfluidic PDMS Chips
			2.3 Polyvinyl Alcohol (PVA) Treatment
			2.4 Generation of Double Emulsion Droplets and Polymerization
			2.5 PEG Treatment
			2.6 Membrane Staining (Optional)
			2.7 DNA Loading
			2.8 Cell-Free Expression (TXTL)
			2.9 Stock Solutions
		3 Methods
			3.1 Fabrication of SU-8 Patterned Silicon Wafers
			3.2 Production of Microfluidic PDMS Chips
			3.3 PVA Treatment
			3.4 Generation of Double Emulsion Droplets and Polymerization
			3.5 PEG Treatment
			3.6 Membrane Staining (Optional)
			3.7 DNA Loading
			3.8 Cell-Free Expression (TXTL)
			3.9 Automatic Image Analysis
		4 Notes
		References
	Chapter 16: Cell-Free Membrane Protein Expression into Hybrid Lipid/Polymer Vesicles
		1 Introduction
		2 Materials
			2.1 Hybrid Vesicle Preparation
			2.2 Vesicle Purification
			2.3 Cell-Free Expression of a GFP-Membrane Protein into Hybrid Vesicles
			2.4 Assessment of Membrane Protein Activity
		3 Methods
			3.1 Hybrid Vesicle Preparation
			3.2 Vesicle Purification
				3.2.1 Purification with an Automated Fraction Collector
				3.2.2 Purification Without the Assistance of a Fraction Collector
			3.3 Cell-Free Expression of a GFP-Membrane Protein into Hybrid Vesicles
			3.4 Assessment of Membrane Protein Activity
		4 Notes
		References
	Chapter 17: Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes
		1 Introduction
		2 Materials
			2.1 Preparation of Liposomes
			2.2 Cell-Free Expression
			2.3 Protein Analysis
		3 Methods
			3.1 Liposome Preparation
			3.2 Cell-Free Expression
			3.3 Sucrose Flotation
			3.4 SDS-PAGE Analysis
			3.5 Protease Digestion of Inserted Protein
			3.6 Pausing and Restarting Translation: Determining How Long It Takes to Make Full-Length Protein
			3.7 Pausing and Restarting Translation
		4 Notes
		References
	Chapter 18: Assembly of RNA Nanostructures from Double-Crossover Tiles
		1 Introduction
		2 Materials
			2.1 RNA Transcription
			2.2 Polyacrylamide Gel Components
			2.3 RNA Extraction, Elution, and Precipitation
			2.4 Assembly Buffer Components
		3 Methods
			3.1 RNA Tile Design
			3.2 DNA and RNA Quantitation
			3.3 Polyacrylamide Gel Electrophoresis (PAGE)
				3.3.1 Denaturing
			3.4 Annealing dsDNA Templates
			3.5 RNA Transcription
			3.6 RNA Purification
			3.7 Assembling RNA Nanotubes by Standard Anneal
			3.8 Assembling RNA Nanotubes by One-Pot Transcription and Anneal
			3.9 Atomic Force Microscopy
			3.10 Fluorescence Microscopy
		4 Notes
		References
	Chapter 19: Cell-Free Biosensors and AI Integration
		1 Introduction
		2 Materials
			2.1 Preparation of Cell-Free Extract and Buffer
			2.2 Cloning of DNA Parts and Production of Plasmids
			2.3 Running Cell-Free Reactions
			2.4 In Silico Materials
		3 Methods
			3.1 Identifying Sensing Routes for a Target Molecule (Fig. 2)
			3.2 Constructing Candidate Biosensors Plasmids
			3.3 Preparing in House Cell-Free Extract and Buffer
				3.3.1 Extract Preparation
				3.3.2 Buffer Preparation
			3.4 Running Cell-Free Reactions
			3.5 Cell-Free Biosensors Characterizations and Optimizations
			3.6 Design, Build, and Test a Perceptron
		4 Notes
		References
	Chapter 20: ROSALIND: Rapid Detection of Chemical Contaminants with In Vitro Transcription Factor-Based Biosensors
		1 Introduction
		2 Materials
			2.1 Preparing a Linear Transcription Template
			2.2 Assembling Fresh ROSALIND Reactions
			2.3 Freeze-Drying ROSALIND Reactions
			2.4 Packaging the Freeze-Dried ROSALIND Reactions
			2.5 Rehydrating the Freeze-Dried ROSALIND Reactions
			2.6 Purifying an Allosteric Transcription Factor, TetR (See Note 10)
		3 Methods
			3.1 Preparing Linear Transcription Template
			3.2 Assembling a 20 μL ROSALIND Reaction for a Microplate Reader Assay
			3.3 Freeze-Drying ROSALIND Reaction
			3.4 Packaging Freeze-Dried ROSALIND Reactions
			3.5 Rehydrating a Freeze-Dried ROSALIND Reaction
			3.6 Purifying an Allosteric Transcription Factor,  TetR
		4 Notes
		References
	Chapter 21: Optical Sensing in Cell-Free Expression
		1 Introduction
		2 Materials
			2.1 Preparation of E. coli Strains Containing YF1/FixJ
			2.2 Preparation of Expression Template
			2.3 Preparation of E. coli Extract Containing YF1/FixJ
			2.4 Preparation of Reaction Mixture
		3 Methods
			3.1 Preparation of E. coli Strains Containing YF1/FixJ
			3.2 Preparation of Expression Template
			3.3 Preparation of E. coli Extract Containing YF1/FixJ
			3.4 Cell-Free Reaction
		4 Notes
		References
	Chapter 22: Cell-Free Paper-Based Analysis of Gut Microbiota and Host Biomarkers
		1 Introduction
		2 Materials
			2.1 Toehold Switch Sensor Assembly and Purification
			2.2 RNA Synthesis and Purification
			2.3 RNA Toehold Switch Cell-Free Assay
			2.4 NASBA (See Note 3)
			2.5 Stool Sample Processing
		3 Methods
			3.1 Identification of Unique Bacterial mRNA Sequences
				3.1.1 Metaphlan In-Group Marker Extraction
				3.1.2 Confirm the Presence of Preliminary Markers in the Target Species (See Note 9)
				3.1.3 Check Marker Expression Levels in Human Stool (See Note 13)
				3.1.4 Assess Marker Specificity (See Note 18)
			3.2 Selection of Host Transcripts
			3.3 RNA Toehold Switch Design
			3.4 Toehold Switch Sensor Assembly and Purification
			3.5 Trigger RNA and mRNA Standard Synthesis
			3.6 Preparation of Paper Substrate
			3.7 Microtiter Plate Setup for RNA Toehold Switch Cell-Free Assay
			3.8 RNA Toehold Switch Cell-Free Assay
			3.9 Analysis of Kinetic Fluorescence  Data
			3.10 RNA Toehold Switch Screening
			3.11 NASBA Reaction
			3.12 NASBA Primer Evaluation
			3.13 Development of NASBA/Toehold Switch Calibration Curve
			3.14 Stool Sample Homogenization and RNA Extraction
		4 Notes
		References
	Chapter 23: Detection of Norovirus Using Paper-Based Cell-Free Systems
		1 Introduction
		2 Materials
			2.1 Preparation of Toehold Switch Plasmids
			2.2 Preparation of Paper-Based Cell-Free Reactions
			2.3 Processing Norovirus Samples, Synbody Enrichment, and Isothermal Amplification
			2.4 Equipment
		3 Methods
			3.1 Preparation of Toehold Switch Plasmids
				3.1.1 Preparing Toehold Switch Insert Sequence
				3.1.2 Preparing Vector Backbones
				3.1.3 Gibson Assembly
				3.1.4 Transformation of Chemically Competent E. coli Cells
				3.1.5 Production of Toehold Switch Plasmid  DNA
			3.2 Preparation of Freeze-Dried Cell-Free Reactions on Paper
				3.2.1 Cell-Free Production of LacZΩ Protein
				3.2.2 Preparation of Paper Substrates
				3.2.3 Preparation of Freeze-Dried Cell-Free Reactions
			3.3 Norovirus Sample Processing and Amplification
				3.3.1 Concentration and Extraction of Norovirus RNA
				3.3.2 NASBA Isothermal Amplification Reactions
			3.4 Paper-Based Cell-Free Detection of Norovirus
		4 Notes
		References
	Chapter 24: A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes
		1 Introduction
		2 Materials
			2.1 Reagents and  Kits
			2.2 Equipment
		3 Methods
			3.1 PAM Library Construction
			3.2 CRISPR-Cas Plasmid Design and Preparation
			3.3 PAM Assay
			3.4 DNA Extraction
			3.5 Quality Check
				3.5.1 Sanger Sequencing
				3.5.2 Quantitative PCR (qPCR)
			3.6 NGS Library Preparation
			3.7 NGS Data Analysis
			3.8 Data Validation
		4 Notes
		References
	Chapter 25: Implementing Hands-On Molecular and Synthetic Biology Education Using Cell-Free Technology
		1 Introduction
		2 Materials
			2.1 Central Dogma Activity
			2.2 Hydrogel Activity
			2.3 Antibiotic Resistance Activity
			2.4 CRISPR Activity
		3 Methods
			3.1 Observing Transcription and Translation in Real  Time
			3.2 Making Biomaterials with Hydrogel-Generating Enzymes
				3.2.1 Hydrogel Monomer Preparation
				3.2.2 Classroom Protocol for Hydrogel Activity
			3.3 Investigating a Mechanism of Antibiotic Resistance
			3.4 Exploring CRISPR-Cas9 Gene Editing
			3.5 Addressing Common Challenges
		4 Notes
		References
Index