Mammalian Synthetic Systems (Methods in Molecular Biology, 2774)

Preface
Contents
Contributors
Chapter 1: A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display
	1 Introduction
	2 Materials
		2.1 DNA Construction and Preparation of Biotinylated Target DNA
		2.2 Preparation of Streptavidin Magnetic Beads
		2.3 In Vitro Transcription and Translation
		2.4 Affinity Selection
	3 Methods
		3.1 Preparation of DNA Templates and Target DNA
		3.2 In Vitro Transcription and Translation
		3.3 Affinity Selection
	4 Notes
	References
Chapter 2: Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems
	1 Introduction
	2 Materials
		2.1 Cloning
		2.2 Screening for Correct Assembled Constructs
	3 Methods
		3.1 Conceptual Design and Consideration of MoPET
		3.2 Design, Generation, and Domestication of Level 0 Functional Parts
		3.3 Design, Generation, and Domestication of Level 1 Expression Plasmids
		3.4 Standard Golden Gate Assembly of Defined Expression Constructs
		3.5 Standard Golden Gate Assembly of Expression Libraries
	4 Notes
	References
Chapter 3: Coiled-Coil Interaction Toolbox for Engineering Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Plasmids
			2.1.1 Equipment
			2.1.2 Reagents
		2.2 Mammalian Cell Culture and Transfection
			2.2.1 Equipment
			2.2.2 Reagents
		2.3 Luciferase Reporter Assay
	3 Methods
		3.1 Selection and Preparation of CC Encoding Plasmids for Inducing PPIs
		3.2 Mammalian Cell Culture and Transfection
		3.3 Determination of Firefly Luciferase Activity
	4 Notes
	References
Chapter 4: A Mammalian-Based Synthetic Biology Toolbox to Engineer Membrane-Membrane Interfaces
	1 Introduction
	2 Materials
		2.1 Cell-Free Reconstitution of sfCherry in Membrane-Membrane Interfaces
			2.1.1 Cell-Free Expression and Protein Dimerization Test
			2.1.2 SUV and SUPER Template Generation
			2.1.3 Bacterial Expression and Purification
			2.1.4 Size Exclusion Chromatography
		2.2 Intercellular sfCherry Reconstitution
			2.2.1 Cell Culturing and Stable Cell Line Generation
	3 Methods
		3.1 Cell-Free Reconstitution of sfCherry in Membrane-Membrane Interfaces
			3.1.1 Cell-Free Expression of sTag and sCatch and Protein Dimerization Test
			3.1.2 SUV and SUPER Template Preparation for Direct Membrane Protein Reconstitution
			3.1.3 Bacterial Expression and Purification of sCatch-GFP-6xHis and sTag-BFP-6xHis
			3.1.4 Size Exclusion Chromatography and Membrane-Membrane Interface Reconstitution
		3.2 Intercellular sfCherry Reconstitution
			3.2.1 Generation of Stable HEK293 Cell Lines Expressing InterCatch-3xL and InterTag-3xL
			3.2.2 Co-Culturing Cells Expressing InterTag-3xL and InterCatch-3xL Protein Systems
	4 Notes
	References
Chapter 5: Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian...
	1 Introduction
	2 Materials
		2.1 In Silico Protein Engineering
		2.2 PCR and Cloning
		2.3 Cell Culture
		2.4 Transient Transfection, Cell Imaging, and Flow Cytometry
	3 Methods
		3.1 Cell Culture
		3.2 Protein Structure Analysis for Re-engineering of RBPs and Plasmid Cloning
			3.2.1 L7Ae
			3.2.2 MS2-cNOT7
		3.3 Test of the Engineered Proteins and Their Interaction with TEV Protease
			3.3.1 Preparation of Transfection Mixes
			3.3.2 Cell Seeding
			3.3.3 Flow Cytometry
	4 Results
	5 Notes
	References
Chapter 6: Mechanistic Model-Driven Biodesign in Mammalian Synthetic Biology
	1 Introduction
	2 Models at the Molecular and Cellular Level
		2.1 Models at the System Level
	3 Next-Generation Models and Methods for Mammalian Cell Synthetic Biology
	4 Challenges and Opportunities
	References
Chapter 7: Realizing Antithetic Integral Feedback Control in Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Plasmid Construction
		2.2 Mammalian Cell Culture
		2.3 Transfection
		2.4 Measurement
		2.5 Data Analysis
	3 Methods
		3.1 Genetic Circuit Assembly
		3.2 Experimental Validation
			3.2.1 Preparation
			3.2.2 Transfections  Plan
			3.2.3 Transfections
			3.2.4 Measurement
			3.2.5 Analysis
		3.3 Characterization of the Steady-State Input/Output Response of the Controlled Network
		3.4 Validation of the Sequestration Reaction
		3.5 Validation of the Integral Feedback
	4 Notes
	References
Chapter 8: A Computational Modeling Approach for the Design of Genetic Control Systems that Respond to Transcriptional Activity
	1 Introduction
	2 Computational Modeling Approach for the Design of Genetic Control Systems
	3 Methods
		3.1 Predictive Mathematical Model
			3.1.1 Modeling Results
			Box 1. Mathematical Model
			3.1.2 Sensitivity Analysis
			3.1.3 Sensitivity Analysis Results
			3.1.4 Parameter Scan
			3.1.5 Parameter Scan Results
	4 Notes
	References
Chapter 9: Flux Balance Analysis of Mammalian Cell Systems
	1 Introduction
	2 Materials
		2.1 Stoichiometric Network Model
		2.2 Input Constraints
		2.3 Software Implementation
	3 Methodology
		3.1 Flux Balance Analysis
		3.2 FBA-Based Methodologies in Mammalian Synthetic Biology
			3.2.1 Strain Design
			3.2.2 Cell Line-Specific Models
		3.3 Flux Sampling
	References
Chapter 10: Multiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Int...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Plasmids and Cell Lines
		2.3 Reagents for Plasmid Generation
		2.4 Reagents for Landing Pad Generation and Plasmid Recombination
		2.5 Reagents for Genomic DNA Extraction and High-Throughput Sequencing Sample  Prep
	3 Methods
		3.1 Production of Lenti-Landing Pad Lentiviral Vector Particles
		3.2 Lentiviral Transduction to Generate Landing Pad Cells
		3.3 Validation of Landing Pad Clonal Lines
		3.4 Recombination Plasmid Generation
		3.5 Plasmid Recombination into Landing Pad Cells
		3.6 Illumina Sequencing of Genomically Integrated  DNA
	4 Notes
	References
Chapter 11: Flow Cytometry Quantification of Transient Transfections in Mammalian Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Data Collection
		3.2 Log-Scale Statistical Analyses
		3.3 Bead-Based Fluorescence Channel Calibration
		3.4 Bead-Based Particle Size Calibration
		3.5 Cell Gating
		3.6 Fluorescence Compensation
		3.7 Fluorescent Channel Conversion
		3.8 Experimental Sample Analysis
	4 Notes
		4.1 Notes on Reagents
			4.1.1 Calibrant Reagents
			4.1.2 Cellular Calibrants
			4.1.3 Experimental Samples
		4.2 Null Transfection Control
		4.3 Data Collection
		4.4 Small Fraction of Cell-Like Particles
		4.5 Alternative Statistics Analyses
		4.6 Bead-Based Fluorescence Channel Calibration
		4.7 Bead-Based Particle Size Calibration
		4.8 Bead Peak Problems
		4.9 Cell Gating
		4.10 Fluorescence Compensation
		4.11 Compensation Problems
		4.12 Channel Conversion Problems
		4.13 Fluorescent Channel Conversion
		4.14 Experimental Sample Analysis
		4.15 Transfection Problems
	References
Chapter 12: RNA Switches Using Cas Proteins
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Plasmid Construction
		2.2 RNA Template and RNA Construction
		2.3 Cell Culture, Plasmid/RNA Transfection, and Assay
	3 Methods
		3.1 Construction of the Cas Protein-Responsive Switch (The Principle of the Switch Is Described in Note 1)
			3.1.1 SpCas9-Responsive Switch (Gluc-Sp_gRNA-EGFP) (Fig. 1 and See Note 2)
		3.2 Construction of the Cas Protein-Responsive Switch and Its Trigger mRNA
			3.2.1 Construction of the SpCas9 mRNA (Trigger), SpCas9-Responsive mRNA (Gluc-Sp_gRNA-EGFP mRNA, Switch), pAptamerCassette-EGF...
			3.2.2 Construction of RNA by In Vitro Transcription (IVT) (See Note 4)
		3.3 Validation
			3.3.1 Preparation of Samples
			3.3.2 Transfection of Plasmid (See Note 5)
			3.3.3 Transfection of mRNA (See Note 6)
			3.3.4 Validation of the Switch Performance
	4 Notes
	References
Chapter 13: Multiplexed Transactivation of Mammalian Cells Using dFnCas12a-VPR
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Consumables
		2.3 Commercial Reagents
	3 Methods
		3.1 Design and Generation of Single crRNA Plasmids
		3.2 Screening Activity of Single crRNAs by qRT-PCR
		3.3 Generation and Testing of Multiplexed Arrays
	4 Notes
	References
Chapter 14: Anti-CRISPR Proteins and Their Application to Control CRISPR Effectors in Mammalian Systems
	1 Introduction
	2 Applications of Anti-CRISPR Proteins
		2.1 Constitutive Expression of Acrs to Protect Cells from CRISPR Gene Editing
		2.2 Timely Delivery of Acrs to Reduce CRISPR Off-Target Editing Activity and Cellular Toxicity
		2.3 Transcriptional and Posttranscriptional Control of Acr Transgenes Facilitates Complex CRISPR Circuits As Well As Cell-Type...
		2.4 Engineered Acr-Based Switches for Light- or Ligand-Controlled CRISPR-Cas Inhibition
		2.5 Employing Acrs for CRISPR-Cas Detection
	3 Conclusions and Future Perspectives
	References
Chapter 15: Posttranslational Remote Control Mediated by Physical Inducers for Rapid Protein Release in Engineered Mammalian C...
	1 Introduction
	2 Materials
		2.1 Construction of a Stable β-Cell Line Co-expressing Insulin-nLuc
		2.2 Cell Culture
		2.3 nLuc Measurement
		2.4 Insulin Measurement
	3 Methods
		3.1 Plate the β-Cell  Line
		3.2 Transfection of Light-Sensitive Receptor into β-Cell  Line
		3.3 Illumination Experiment
		3.4 nLuc Secretion Measurement
		3.5 Insulin Secretion Measurement
	4 Notes
	References
Chapter 16: Detection of MicroRNAs Using Synthetic Toehold Switch in Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Plasmids
		2.2 Materials
			2.2.1 Cloning Materials
			2.2.2 Cell Culture Material
			2.2.3 Transfection
			2.2.4 Reverse Transcription and RT-PCR
		2.3 Equipment
	3 Methods
		3.1 Construction of Toehold Structures
		3.2 Plasmid Construction (See Note 2)
			3.2.1 Construct Toehold Switch for miR-155 Detection
			3.2.2 Construct Toehold Switch for miR-21 Detection
			3.2.3 Construct Control Toehold Switch
		3.3 Cell Culture and Transfection
		3.4 Reverse Transcription and RT-PCR
			3.4.1 Cell Lysis
			3.4.2 Reverse Transcription
			3.4.3 Real-Time PCR
			3.4.4 Data Analysis
		3.5 Flow Cytometry and Toehold Switch Characterization
		3.6 Detection of Exogenously Expressed miRNAs
		3.7 Detection of Simulated Endogenous miRNAs
		3.8 Multiplex Detection of miRNAs
	4 Notes
	References
Chapter 17: Imaging S-Adenosyl Methionine Dynamics in Living Cells Using an RNA-Based Fluorescent Sensor
	1 Introduction
	2 Materials
		2.1 Cell Culture Reagents
		2.2 Transfection Reagents
		2.3 Imaging Reagents
	3 Methods
		3.1 Culturing and Seeding Cells
		3.2 Transfection
		3.3 Imaging
		3.4 Data Analysis
	4 Notes
	References
Chapter 18: High-Throughput Spectroscopic Analysis of mRNA Capping Level
	1 Introduction
	2 Materials and Reagents
		2.1 In Vitro mRNA Synthesis, Capping, and Polyadenylation
		2.2 mRNA Substrates and Reporter Enzymes
			2.2.1 Materials
			2.2.2 Reagents
		2.3 Cell Culture
			2.3.1 Materials
			2.3.2 Reagents
		2.4 Transfection of mRNAs into HEK293T Mammalian Cells
			2.4.1 Reagents
		2.5 Assessment of Expression by Flow Cytometry
			2.5.1 Materials
			2.5.2 Reagents
	3 Methods
		3.1 In Vitro mRNA Transcription
		3.2 mRNA Purification
		3.3 Rehydrating Biotinylated Poly-deoxythymidine (pdT25)
		3.4 Equilibration of Streptavidin-Coated Magnetic Beads and Immobilization with pdT25
		3.5 mRNA Capture by pdT25
		3.6 B4E Binding to Capped  mRNA
		3.7 Nitrocefin Colorimetric Assay
		3.8 Cell Culture
		3.9 mRNA Transfection
		3.10 Assessment of Protein Expression by Flow Cytometry
	4 Notes
	References
Chapter 19: In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells
	1 Introduction
	2 Materials
		2.1 Expanding and Mitomycin C Treating Mouse Embryonic Fibroblasts (MEFs)
		2.2 Expanding Mouse Embryonic Stem Cells (mESCs)
		2.3 Expanding OP9 Cells
		2.4 Differentiating mESCs
		2.5 Docking Transgenes into the Genome of mESCs Using 훟C31 Integrase
		2.6 Flow Cytometry Analysis of Differentiation and Loaded Transgene Markers
		2.7 Imaging for Analysis of Differentiation and Loaded Transgene Markers
	3 Methods
		3.1 Expanding and Mitomycin C Treating  MEFs
		3.2 Expanding Mouse Embryonic Stem Cells (mESCs)
		3.3 Expanding OP9 Cells and Plating for mESC Differentiation Studies
		3.4 Differentiating mESCs
			3.4.1 Preparing OP9 Cells (Fig. 4)
			3.4.2 Differentiating mESCs (Fig. 5)
		3.5 Docking Transgenes into the Genome of mESCs Using 훟C31 Integrase
		3.6 Flow Cytometry
		3.7 Imaging
	4 Notes
	References
Chapter 20: Preparation of Chromobodies for the Detection of Cell Surface Epitopes
	1 Introduction
	2 Materials
		2.1 Mammalian Cell Lines and Plasmids
		2.2 Mammalian Cell Media
		2.3 Transfection Reagents
		2.4 Molecular Cloning
		2.5 Immunofluorescence for Analysis
	3 Methods
		3.1 Cloning of Nanobody CDS into Expression Plasmid
		3.2 Transfection and Chromobodies Production
		3.3 Detecting Surface Epitopes Using Chromobodies via Microscopy
		3.4 Detecting Surface Epitopes Using Chromobodies via Flow Cytometry
	4 Notes
	References
Index