Synthetic Biology: Methods and Protocols (Methods in Molecular Biology, 2760)

Preface
Contents
Contributors
Part I: Gene Circuits and Biochemical Pathways
	Chapter 1: Plant Engineering to Enable Platforms for Sustainable Bioproduction of Terpenoids
		1 Introduction
		2 Terpenoid Production in Plants
		3 Engineering Strategies to Improve the Bioproduction of Terpenoids in Plants
		4 Compartmentalization of Pathways
		5 Engineering Plants for the Production of Terpenoids
		6 Advancements in Plant Genetic Engineering
		7 Conclusions
		References
	Chapter 2: Compartmentalized Terpenoid Production in Plants Using Agrobacterium-Mediated Transient Expression
		1 Introduction
		2 Materials
			2.1 Vector Assembly
			2.2 Preparation and Transformation of Agrobacterium Competent Cells
			2.3 N. benthamiana Transformation
		3 Methods
			3.1 Constructing Transient Expression Vectors
			3.2 Preparation and Transformation of Agrobacterium Competent Cells
			3.3 Syringe Infiltration
			3.4 Vacuum Infiltration
			3.5 Analytics
		4 Notes
		References
	Chapter 3: Design Principles for Biological Adaptation: A Systems and Control-Theoretic Treatment
		1 Introduction
		2 Mathematical Preliminaries
			2.1 Systems Theory: A Primer
			2.2 Relevant Results on Algebraic Graph Theory
		3 Methodology and Applications
			3.1 Proposed Methodology
			3.2 Identifying Adaptive Network Structures
				3.2.1 Assumptions
			3.3 Conditions for Adaptation
			3.4 From Abstraction to Structure
		4 Conclusions
		5 Notes
		References
	Chapter 4: Construction of Xylose-Utilizing Cyanobacterial Chassis for Bioproduction Under Photomixotrophic Conditions
		1 Introduction
		2 Materials and Equipment
			2.1 Target Genes, Plasmids, and Strains
			2.2 Medium
			2.3 Chemicals and Reagents
			2.4 Sample Content Analysis
			2.5 Equipment
		3 Methods
			3.1 Determination of Xylose-Utilizing Capability of Wild-Type Synechococcus 2973
			3.2 Construction of Xylose-Utilization Pathway in Synechococcus 2973
			3.3 Determination of Xylose Content During Photomixotrophic Conditions
			3.4 Determining the Efficiency of Converting Xylose to Intracellular Acetyl-CoA in Strain JQ01
			3.5 Analyze the Metabolism Changes of Strain JQ01 Under Photomixotrophic Conditions
			3.6 Drive More Carbon Flux to Acetyl-CoA by Rewiring Central Carbon Metabolism
			3.7 Bioproduction of 3-HP in the Rewired Chassis Under Photomixotrophic Conditions
		4 Notes
		References
	Chapter 5: Allosteric-Regulation-Based DNA Circuits in Saccharomyces cerevisiae to Detect Organic Acids and Monitor Hydrocarbo...
		1 Introduction
		2 Materials
			2.1 In Silico DNA Analysis and PCR
			2.2 Agarose Gel Electrophoresis
			2.3 Isothermal DNA Assembly (Gibson Method)
			2.4 Yeast Transformation
			2.5 Media: Solutions and Plates
			2.6 Fluorescence-Activated Cell Sorting (FACS)
			2.7 Other Chemicals and Reagents
		3 Methods
			3.1 Biosensor Design and Assembly
			3.2 Touchdown PCR
			3.3 Bacterial Transformation and Sequence Verification
			3.4 Integration into the Genome of S. cerevisiae
			3.5 Cell Selection-Reporter Element
			3.6 Cell Selection-Complete Circuit (Receptor and Reporter)
			3.7 FACS Measurements
			3.8 Detection of Putative Compounds in Environmental Samples
			3.9 Real-Time Monitoring of Hydrocarbon Metabolism
		4 Notes
		References
	Chapter 6: dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation in Saccharomyces cerevisiae Synthetic Gene Circuits
		1 Introduction
		2 Materials
			2.1 PCR
			2.2 Agarose Gel Electrophoresis
			2.3 Agarose Gel Preparation
			2.4 Gibson Reaction Buffer
			2.5 Gibson Assembly Master Mixture
			2.6 Mini-preparation
			2.7 Yeast Transformation
			2.8 FACS Beads and Cleaning Solution Preparation
			2.9 Media and Plate
		3 Methods
			3.1 Plasmid Design
			3.2 Gene Circuit Design
			3.3 Touchdown PCR
			3.4 Agarose Gel Electrophoresis
			3.5 Backbone Digestion
			3.6 Gibson Assembly
			3.7 Escherichia coli DH5α Transformation
			3.8 Mini-preparation
			3.9 Checking the Sequences of the Assembled Plasmids
			3.10 Yeast Transformation
			3.11 Strain Selection: FACS Measurement
			3.12 Strains Transformed with Two Plasmids
			3.13 Strains Transformed with the Complete IMPLY Boolean Gates Responding to Copper and Methionine
		4 Notes
		References
Part II: Genome Editing and Modification
	Chapter 7: CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes
		1 Introduction
		2 Materials
			2.1 Synthesis of Oligomer Llibrary and Assembly of Plasmid Library
			2.2 Transformation of sgRNA Plasmid Library to Helper Strain for Conjugation
			2.3 Conjugation of Bacterial Hosts
			2.4 Quality Control (QC) of CRISPRi Library
			2.5 Growth Profiling of CRISPRi Library to Provide Selected Library
		3 Methods
			3.1 Design Considerations for sgRNA Oligomer Library
			3.2 Synthesis of Oligomer Library and Assembly of Plasmid Library
				3.2.1 Assembly of Plasmid Libraries
			3.3 Transformation of sgRNA Plasmid Library to Helper Strain for Conjugation
				3.3.1 Preparation of Electrocompetent Cells
				3.3.2 Electroporation
				3.3.3 Transformation of sgRNA Plasmid Library to Helper Strain
			3.4 Conjugation of Bacterial Hosts
			3.5 Quality Control (QC) of CRISPRi Library
				3.5.1 Sequencing Library Preparation
				3.5.2 Running  NGS
				3.5.3 Sequencing Analysis
			3.6 Determination of Gene Essentiality
				3.6.1 Growth Profiling of CRISPRi Library to Provide Selected Library
				3.6.2 Calculation of Gene Fitness Score
				3.6.3 Identification of Essential Genes
		4 Notes
		References
	Chapter 8: Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its ...
		1 Introduction
		2 Materials
			2.1 Reagents, Kits, and Apparatus
			2.2 Oligonucleotides
		3 Methods
			3.1 Primer Extension with Unnatural Nucleoside Triphosphates by TdT
			3.2 Enzyme-Linked Oligonucleotide Assay (ELONA) with a dTPT3Bio-Labeled DNA Aptamer
			3.3 Imaging of Bacterial Cells with a dTPT3FAM-Labeled DNA Aptamer
			3.4 Enzymatic Synthesis of DNA Strands Containing an Internal UB/UBP
				3.4.1 Preparation of DNA Oligonucleotides Containing an Internal Unnatural Nucleobase
				3.4.2 PCR Amplification of the Unnatural Nucleobase-Containing DNA Oligonucleotide Product and Biotin Gel Shift Assay
				3.4.3 Magnetic Beads Purification of the UBP-Containing DNA Product Produced by PCR
		4 Notes
		References
	Chapter 9: Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a
		1 Introduction
		2 Materials
			2.1 Electroporation
			2.2 MacConkey  Agar
			2.3 Sanger Sequencing
		3 Methods
			3.1 Preparation of Electrocompetent  Cell
			3.2 Electroporation
			3.3 Sanger Sequencing
		4 Notes
		References
	Chapter 10: Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9
		1 Introduction
		2 Materials
			2.1 Gibson Assembly (GA) Mix
			2.2 Buffer and Reagents
			2.3 Culture Medium
		3 Methods
			3.1 Plasmid Construction
			3.2 Transformation of P. pastoris Competent Cells
			3.3 Validation of P. pastoris Transformants
		4 Notes
		References
	Chapter 11: Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia ...
		1 Introduction
		2 Materials
			2.1 Molecular Biology Reagents
			2.2 Cell Culture and Transformation
		3 Methods
			3.1 Design and Cloning of Gene Disruption Constructs
			3.2 Transformation of Y. lipolytica with CRISPR-Cas12a Plasmids for Genome Editing or Transcriptional Regulation
			3.3 Genome Mutation Analysis
			3.4 Design, Cloning, and Application of CRISPRi (Interference) and CRISPRa (Activation) Using CRISPR-Cas12a
			3.5 Design, Cloning, and Validation of a Genome-Wide CRISPR-Cas12a Library for Pooled CRISPR Screening in Y. lipolytica
			3.6 Pooled CRISPR Screening Protocol for Genome-Wide Cas12a gRNA Activity Profiling in Y. lipolytica
			3.7 Bioinformatic Processing of NextSeq Reads to Obtain gRNA Abundances
		4 Notes
		References
	Chapter 12: An Improved Method for Eliminating or Creating Intragenic Bacterial Promoters
		1 Introduction
		2 Materials
			2.1 Hardware and Software Requirements
				2.1.1 Software
		3 Methods
			3.1 Choose Coding Sequence
			3.2 Generate New Sequences with σ70 Promoters Weakened (CORPSE) or Strengthened (iCORPSE)
			3.3 Analyzed Results
		4 Notes
		References
	Chapter 13: Genetic Code Expansion in Pseudomonas putida KT2440
		1 Introduction
		2 Materials
			2.1 Media and Reagents
			2.2 Plasmids
			2.3 Equipment
		3 Methods
			3.1 Cell Cultivation
			3.2 Competent Cells Preparation (See Note 5)
			3.3 Heat Shock Transformation of Plasmids
			3.4 Fluorescence Measurement
			3.5 Purification of sfGFP Variant Proteins
			3.6 Sample Preparation for MS Analysis of Purified sfGFP
		4 Notes
		References
	Chapter 14: Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast
		1 Introduction
		2 Materials
			2.1 Transformation of Reporter System into Yeast
			2.2 Prepare Cells for Electroporation
			2.3 Electroporation
			2.4 Yeast Culture Expansion
			2.5 Long-Term Library Storage
			2.6 Library Characterization
			2.7 Sanger Sequencing
			2.8 Fluorescence-Activated Cell Sorting (FACS)
			2.9 Deep Sequencing
		3 Methods
			3.1 Transform pSPS-RepTAG-OTS and pSPS-Rep-OTS into Chemically Competent Yeast
			3.2 Prepare Cells for Electroporation
				3.2.1 Yeast Culture Growth
				3.2.2 Pellet Paint
				3.2.3 Making Electrocompetent Cells
			3.3 Electroporation
			3.4 Expanding Culture
			3.5 Long-Term Library Storage
			3.6 Library Characterization
				3.6.1 Prepare Cells for Flow Cytometry Analysis
				3.6.2 Flow Cytometry
			3.7 Sequencing Clones Isolated from Naïve Library
			3.8 Fluorescence-Activated Cell Sorting
				3.8.1 Analytical Flow Cytometry on Recovered Sorts
				3.8.2 Sorted Library Characterization
			3.9 Deep Sequencing
		4 Notes
		References
	Chapter 15: Positive Selection Screens for Programmable Endonuclease Activity Using I-SceI
		1 Introduction
		2 Materials
			2.1 Cell Culture and Cultivation
			2.2 Molecular Biology Reagents
		3 Methods
			3.1 Electrocompetent Cell Creation
			3.2 Transformation (Electroporation)
			3.3 I-SceI Lethality Validation (Fig. 3)
			3.4 Nuclease Activity Assay (Fig. 4)
			3.5 Enrichment and Positive Selection (Fig. 6)
		4 Notes
		References
	Chapter 16: CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa
		1 Introduction
		2 Materials
			2.1 Molecular Biology
			2.2 Cultivation and Transformation
		3 Methods
			3.1 Cloning of Plasmids for Deletions and Integrations
			3.2 Chemical Transformation of E. coli TOP10, E. coli Turbo, and E. coli S17-1
			3.3 Conjugation
			3.4 Multipurpose Genome Editing
				3.4.1 Deletion and Integration
				3.4.2 Point Mutations
				3.4.3 Multiplexing
		4 Notes
		References
Part III: Genome Language and Computing
	Chapter 17: Programming Juxtacrine-Based Synthetic Signaling Networks in a Cellular Potts Framework
		1 Introduction
		2 Materials
		3 Methods
			3.1 Getting Started
			3.2 Creating Cells in CC3D
			3.3 Adding Adhesion to the In Silico Cell Line (ISCL)
			3.4 Adding Motility to ISCL
			3.5 Adding Size, Growth, and Division to ISCL
			3.6 Adding Genetic Circuits to ISCL: Assigning and Tracking Reporter Levels in Cells
			3.7 Adding Genetic Circuits to ISCL: Defining the Signal from Ligands on Neighbor Cells
			3.8 Adding Genetic Circuits to ISCL: Determining How the Focal Cell Responds to Signal from Ligands on Neighbors
			3.9 Adding Genetic Circuits to ISCL: Changing the State of the Cell as a Result of Reporter Accumulation
			3.10 Setting the Initial Simulation State
			3.11 Technical Simulation Parameters
			3.12 Running the Simulation
			3.13 Optional: Quantifications for Monitoring the Signaling Network
			3.14 Optional: Parameterscan
		4 Notes
		References
	Chapter 18: Encoding Genetic Circuits with DNA Barcodes Paves the Way for High-Throughput Profiling of Dose-Response Curves of...
		1 Introduction
		2 Materials
			2.1 Synthetic Complete Medium Minus Ura (SC-Ura)
			2.2 Yeast Transformation Buffer
		3 Methods
			3.1 Trackable Assembly Methods
			3.2 Transformation of Plasmids into Yeast
			3.3 Sort-Seq Experiment
			3.4 NGS Library Preparation
			3.5 Data Processing and Machine Learning
		4 Notes
		References
	Chapter 19: Machine Learning for Biological Design
		1 Introduction
			1.1 Predictive Models and Supervised Learning
			1.2 Nomenclature
			1.3 Anatomy of an Adaptive Experimental Design Workflow
		2 Different Design Objectives
			2.1 A Working Example
		3 Action Improvement
			3.1 Bayesian Optimization
			3.2 Bandits
			3.3 Reinforcement Learning
			3.4 Recommendations and Further Reading
		4 Predictor Improvement
			4.1 Bayes Optimal Experiment Design
			4.2 Model Discrimination
			4.3 Active Learning
			4.4 Recommendations and Further Reading
		5 Discussion and Conclusion
			5.1 Greedy, Non-Greedy, and Batch Algorithms
			5.2 Summary
		References
	Chapter 20: A Machine Learning Approach for Predicting Essentiality of Metabolic Genes
		1 Introduction
		2 Theoretical Background and Concepts
			2.1 Genome-Scale Metabolic Models
			2.2 Flux Balance Analysis
			2.3 Prediction of Gene Essentiality with FBA
			2.4 Mass Flow Graphs
		3 Binary Classification
		4 Gene Essentiality Data for Model Training
			4.1 Mass Flow Graph of iML1515
			4.2 Feature Extraction
			4.3 Essentiality Labels
				4.3.1 Algorithm for Gene to Reaction Essentiality Mapping
		5 Binary Classifiers Trained on Mass Flow Graphs
			5.1 Data Standardisation
			5.2 Dimensionality Reduction
			5.3 Evaluating Classification Performance
			5.4 Application to Escherichia Coli Metabolic Network
				5.4.1 Baseline Classification Models
				5.4.2 Hyperparameter Tuning
				5.4.3 Dimensionality Reduction using Principle Component Analysis
				5.4.4 Evaluation on the Test Set
		6 Transfer Learning
		7 Discussion
		Appendix 1: Acronyms
		Appendix 2: Mapping Gene Essentiality to Reaction Essentiality
		Appendix 3: List of Genes in the Test Set
		References
	Chapter 21: The Causes for Genomic Instability and How to Try and Reduce Them Through Rational Design of Synthetic DNA
		1 Introduction
			1.1 SSR and RMD in Genetic Instability
		2 Counter Selection for SSR in Evolutionary Conserved Sequences
		3 ESO Detection of SSR and RMD Sites
		4 Epigenetics and the Challenges It Presents to Synthetic Biology
		5 ESO Detection of Methylation Motifs
			5.1 ESO Automatic DNA Sequence Optimization
		6 Conclusions
		Bibliography
	Chapter 22: Genetic Network Design Automation with LOICA
		1 Introduction
		2 Materials
			2.1 Dependencies
			2.2 Installation
		3 Methods
			3.1 Designing a NOR Gate
			3.2 Genetic Ring Oscillator
			3.3 Receiver and Inverter Characterization
		4 Notes
		References
	Chapter 23: Flapjack: Data Management and Analysis for Genetic Circuit Characterization
		1 Introduction
		2 Materials
			2.1 Installation
		3 Methods
			3.1 Data Model
			3.2 Accessing Flapjack
				3.2.1 Creating an Account
				3.2.2 Logging in to an Existing Account
			3.3 Home  Page
			3.4 Preparing and Uploading Data in Flapjack
				3.4.1 Preparing the Data  File
				3.4.2 Uploading the  File
					Study
						Creating a New Study
					Machine
					Data  File
			3.5 Browse  Page
				3.5.1 Studies
					Actions
						Share
						Make Public
						Delete
				3.5.2 Assays
				3.5.3 Vectors
			3.6 View  Page
				3.6.1 View Page Filters and Options
					Query
					Analysis
					Plot Options
			3.7 Creating a  Plot
				3.7.1 Induction Curve
			3.8 pyFlapjack
				3.8.1 Importing pyFlapjack
				3.8.2 Connecting to Flapjack
				3.8.3 Functions
					get Function
					Create Function
					Delete Function
					Analyzing  Data
		4 Notes
			4.1 Patch Function
		References
Part IV: Molecular Assembly
	Chapter 24: In Vivo DNA Assembly Using the PEDA Method
		1 Introduction
		2 Materials
			2.1 Polymerase Chain Reaction (PCR)
				2.1.1 Equipment
				2.1.2 Reagents
			2.2 DNA Electrophoresis and Purification
				2.2.1 Equipment
				2.2.2 Reagents
			2.3 Electrocompetent E. coli Cells
				2.3.1 Equipment
				2.3.2 Reagents
		3 Methods
			3.1 Design the Primers
			3.2 Amplification of the DNA Fragments
				3.2.1 Prepare the Following Master Mixture in 50 μL Volume
				3.2.2 Set Up the Thermal Cycling Under the Following Program
			3.3 Purification of the DNA Fragments
			3.4 Preparation of the Electrocompetent Cells
			3.5 Electrotransformation of DNA into the Cells
			3.6 Validation of the Assembled DNA by Colony PCR
		4 Notes
		References
	Chapter 25: Cell-Free Synthesis and Quantitation of Bacteriophages
		1 Introduction
		2 Materials
			2.1 Phage Amplification
			2.2 Phage DNA Extraction and Purification
			2.3 Cell-Free Gene Expression
			2.4 Dilutions of Cell-Free Synthesized Phage Reactions
			2.5 Spotting Assay
			2.6 Kinetic Assay
		3 Methods
			3.1 Preparation of a Phage Lysate
			3.2 Phage DNA Extraction
			3.3 TXTL of Phages
			3.4 Serial Dilutions of Cell-Free Synthesized Phages
			3.5 Phage-Host Infection Spotting Assay
			3.6 Phage-Host Infection Kinetics Assay
		4 Notes
		References
	Chapter 26: Multimodal Control of Bacterial Gene Expression by Red and Blue Light
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Cultivation
			2.2 Implementation and Analysis of Individual Plasmid Systems
				2.2.1 pREDusk and pREDawn
				2.2.2 pCrepusculo and pAurora
			2.3 Multiplexing of Systems
			2.4 Multiplexed Optogenetic Control of Semi-preparative Protein Production
		3 Methods
			3.1 Implementation and Analysis of Individual Plasmid Systems
				3.1.1 pREDusk and pREDawn
				3.1.2 pAurora and pCrepusculo
			3.2 Multiplexing of Systems
			3.3 Multiplexed Optogenetic Control of Semi-preparative Protein Production
		4 Notes
		References
	Chapter 27: In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria
		1 Introduction
		2 Materials
			2.1 Computational Equipment
			2.2 Plasmids
			2.3 DNA Oligonucleotides
			2.4 Enzymes
			2.5 Antibiotics
			2.6 Chemicals, Buffers, and Media Components
			2.7 Consumables
			2.8 Strains
		3 Methods
			3.1 Seed Prediction Using SEEDling
			3.2 Construction and Validation of Synthetic sRNA TUs by Golden Gate Cloning
				3.2.1 Golden Gate Cloning
				3.2.2 Transformation of Golden Gate Reaction into E. coli Cells
				3.2.3 Screening and Validation of Plasmid DNA
			3.3 Synthetic sRNA Functionality Test on Solid Media
			3.4 Synthetic sRNA Functionality Test in Liquid Media
			3.5 Fluorescence Reporter Assay
			3.6 Summary & Perspectives
		4 Notes
		References
Index