Plant Synthetic Biology: Methods and Protocols (Methods in Molecular Biology, 2379)

Preface
Acknowledgments
Contents
Contributors
Chapter 1: Cas9-Mediated Targeted Mutagenesis in Plants
	1 Introduction
	2 Materials
		2.1 Selection of sgRNAs and Design of Plasmid Constructs
		2.2 Assembly of Plasmid Constructs
		2.3 Assessing the Mutagenesis Activity of Cas9-sgRNA Constructs Using Transient Expression
	3 Methods
		3.1 Selection of sgRNAs and Design of Plasmid Constructs
		3.2 Assembly of Plasmid Constructs
		3.3 Assessing the Mutagenesis Activity of Cas9-sgRNA Constructs Using Transient Expression
			3.3.1 Transient Expression in N. benthamiana Leaves via Agroinfiltration
			3.3.2 Transient Expression in Protoplasts
		3.4 Assessment of Targeted Mutagenesis Following Transient Transfection
			3.4.1 Detection of Indels Induced Using a Single sgRNA by PCR and Restriction Endonuclease Digestion (PCR-REN) and Sanger Sequ...
			3.4.2 Detection of Indels Induced Using Paired sgRNAs, PCR, and Sanger Sequencing
		3.5 Production of Stable Lines with Targeted Mutations
	4 Notes
	References
Chapter 2: Design of Multiplexing CRISPR/Cas9 Constructs for Plant Genome Engineering Using the GoldenBraid DNA Assembly Stand...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Choice of the Gene(s) of Interest for Editing, Activation, or Repression
		3.2 Considerations Concerning the Selection of Target Sites
		3.3 Guide RNAs Design for Multiplexing with Goldenbraid
		3.4 Cloning of Level 0 tRNA-Protospacer-Scaffold ``tps´´ Parts
		3.5 Level 1 Polycistronic gRNA Cassette Assembly
		3.6 Final T-DNA Expression Vector Assembly by Combining the Polycistronic gRNA with the dCas9 TU
		3.7 Transient Expression in N. benthamiana Leaves
		3.8 Functional Characterization of the Generated Plasmid for the nos Promoter Regulation
		3.9 Functional Validation of the Generated Plasmid for XT Gene Editing
	4 Notes
	References
Chapter 3: Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins
	1 Introduction
	2 Materials
		2.1 Algal Transformation
		2.2 PCR-Based Mutant Screening
	3 Methods
		3.1 Experimental Design and Pretest for Cas9 Activity
		3.2 Electroporation of Algae Cells with Cas9 RNPs
		3.3 Screening for Mutants
	4 Notes
	References
Chapter 4: pSHDY: A New Tool for Genetic Engineering of Cyanobacteria
	1 Introduction
	2 Materials
		2.1 Microorganisms Used
		2.2 Cloning of Constructs of Interest Using the pSHDY Plasmid
		2.3 Triparental Mating in Synechocystis sp. PCC 6803
		2.4 Culturing of Cyanobacteria for Plate Reader Measurements
	3 Methods
		3.1 Design and Cloning of Constructs of Interest Using pSHDY
		3.2 Triparental Mating of Synechocystis sp. PCC 6803
		3.3 Culturing of Cyanobacteria for Microplate Reader Measurements
	4 Notes
	References
Chapter 5: Plant X-tender Toolbox for the Assembly and Expression of Multiple Transcriptional Units in Plants
	1 Introduction
	2 Materials
		2.1 Designing the Constructs and Primers
		2.2 Cloning
		2.3 Testing Functionality of the Assembled Transcriptional Units
	3 Methods
		3.1 Using GenoCAD to Design a Single Transcriptional  Unit
		3.2 Primer Design
		3.3 Cloning into Level 0 Vectors
		3.4 Cloning into Level 1 Vectors
		3.5 Cloning into Plant X-tender Expression Vectors
		3.6 Testing Functionality of the Assembled Transcriptional Units
	4 Notes
	References
Chapter 6: Exploiting the Gal4/UAS System as Plant Orthogonal Molecular Toolbox to Control Reporter Expression in Arabidopsis ...
	1 Introduction
	2 Materials
		2.1 Reporter and Effector Plasmids
		2.2 Maxi-Preparation of Plasmid  DNA
		2.3 Protoplast Isolation and Transformation
		2.4 Dual Luciferase Reporter Assay (See Note 1)
	3 Methods
		3.1 Cloning of Reporter and Effector Plasmids
		3.2 Maxi-Preparation of High-Quality Plasmid DNA for Protoplast Transformation
		3.3 Protoplast Isolation and Transformation
		3.4 Dual Luciferase Assay Activity
	4 Notes
	References
Chapter 7: AGROBEST: A Highly Efficient Agrobacterium-Mediated Transient Expression System in Arabidopsis Seedlings
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Agrobacterium Strain and Vector
		2.3 Medium for Co-culture of Arabidopsis Seedlings and Agrobacterium
		2.4 Preparation of Agrobacterium Containing T-DNA Binary Vector by Electroporation
		2.5 Preparation of Agrobacterium Culture for Infection
		2.6 Preparation of Arabidopsis Seedlings for Transformation
		2.7 Co-cultivation and Infection
		2.8 GUS Staining Stock Solution
	3 Methods
		3.1 Preparation of Agrobacterium Containing T-DNA Binary Vector
		3.2 Preparation of Agrobacterium Culture for Infection
		3.3 Preparation of Arabidopsis Seedlings for Transformation
		3.4 Co-cultivation and Infection
		3.5 GUS Staining
	4 Notes
	References
Chapter 8: Heterologous Production of Plant Terpenes in the Photosynthetic Bacterium Rhodobacter capsulatus
	1 Introduction
	2 Materials
		2.1 Generation of Gene Sequences for Expression
		2.2 Cloning of Expression Vectors
		2.3 Cultivation of R. capsulatus for Recombinant Terpene Production
		2.4 n-Dodecane Sampling and GC Analysis of Produced Terpenes
		2.5 Cell Extraction and LC Analysis of Produced Terpenes
	3 Methods
		3.1 Generation of Gene Sequences for Expression
		3.2 Cloning of Expression Vectors
			3.2.1 Insertion of a Terpene Synthase Gene in Expression Vectors with Precursor Biosynthetic Genes
			3.2.2 Insertion of an OSC Gene in an Expression Vector with Precursor Biosynthetic Genes for Cyclic Triterpene Biosynthesis
		3.3 Cultivation of R. capsulatus for Recombinant Terpene Production
		3.4 Sample Preparation for Analysis of Produced Terpenes
			3.4.1 Sampling of an n-Dodecane Phase Overlaying an R. capsulatus Expression Culture for GC Analysis
			3.4.2 General Considerations Regarding GC Analysis
			3.4.3 Cell Harvesting and Extraction of Terpenes for LC Analysis
			3.4.4 General Considerations Regarding LC Analysis
	4 Notes
	References
Chapter 9: Coexpression and Reconstitution of Enzymatic Cascades in Bacteria Using UbiGate
	1 Introduction
	2 Materials
		2.1 Polymerase Chain Reaction
		2.2 Restriction-Ligation
		2.3 Preparation of Chemically Competent Cells
		2.4 Screening of Colonies
		2.5 Protein Expression and Purification
	3 Methods
		3.1 Amplification of GOI by PCR
			3.1.1 Domestication
			3.1.2 PCR
		3.2 Cloning of GOI into Level 0 Vector
		3.3 Transformation
		3.4 Generation of Positional Level 1 Vectors
		3.5 Assembly of Polycistronic Level 2 Vectors
		3.6 Expression of Recombinant Proteins
		3.7 Preparation of Chemically Competent E. coli Cells
	4 Notes
	References
Chapter 10: Engineering Destabilizing N-Termini in Plastids
	1 Introduction
	2 Materials
		2.1 General Equipment
		2.2 Cloning of the Reporter Construct
		2.3 Plant Transformation and Selection of Transformants
		2.4 Protein Extraction
		2.5 Determination of Protein Concentration
		2.6 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		2.7 Western Blotting and Immunodetection
	3 Methods
		3.1 Cloning of the Reporter Construct
		3.2 Plant Transformation and Selection of Transformants
		3.3 Protein Extraction
		3.4 Determination of Protein Concentration
		3.5 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		3.6 Western Blotting and Immunodetection
	4 Notes
	References
Chapter 11: Genetically Encoded Biosensors for the Quantitative Analysis of Auxin Dynamics in Plant Cells
	1 Introduction
	2 Materials
		2.1 Reagents, Consumables, and  Kits
		2.2 Protoplast Isolation and Transformation
		2.3 Hormone Induction and Luminescence Determination
		2.4 Reagent Setup
		2.5 Equipment
	3 Methods
		3.1 Plant Material Preparation: Estimated Duration: 0.5 h in the Morning and 0.5 h in the Afternoon (Depending on the Amount o...
		3.2 Protoplast Isolation: Estimated Duration: 0.5-1 h (Day 1) and 3-3.5 h (Day 2)
		3.3 Protoplast Transformation: Estimated Duration 1 h (Day 2)
		3.4 Hormone Induction: Estimated Duration 0.5-1 h and 2-6 h Incubation Time
		3.5 Luminescence Determination
		3.6 Statistical Analysis
	4 Aux/IAAs Show Different Sensitivities Toward  IAA
	5 Notes
	References
Chapter 12: Homo-FRET Imaging to Study Protein-Protein Interaction and Complex Formation in Plants
	1 Introduction
		1.1 Förster Resonance Energy Transfer (FRET)
		1.2 Homo-FRET
		1.3 Prerequisites for Fluorescence Anisotropy Measurements
		1.4 What Affects the Polarization of the Fluorescence Signal?
	2 Materials
	3 Methods
		3.1 For a Complete Data Set, the Following Samples Should Be Measured
		3.2 Preparation of Plant Material
		3.3 Calibration Measurements for Anisotropy
		3.4 Homo-FRET Imaging Measurements
		3.5 Potential Pitfalls and Points of Consideration
	4 Notes
	References
Chapter 13: Mathematical Modelling in Plant Synthetic Biology
	1 Introduction
	2 Mathematical Approaches
		2.1 Dynamics
		2.2 Dynamics Across Space
			2.2.1 Trichome Patterning Model
		2.3 Stochastic Models
		2.4 Model-Based Design
	3 Parameter Inference
		3.1 Parameter Identifiability
		3.2 Optimal Experimental Design
	4 Model Selection
		4.1 Likelihood Tests
		4.2 Information Criteria
	5 Model Analysis
		5.1 Phase Plane Analysis
		5.2 Stability Analysis
		5.3 Bifurcation Analysis
		5.4 Sensitivity Analysis
	6 Handling Uncertainty
		6.1 Monte Carlo Methods
		6.2 Spectral Methods
	7 Summary
	References
Chapter 14: In Vivo Epitope Tagging of Plant Mitochondria
	1 Introduction
	2 Materials
		2.1 Cloning of Tagging Construct
		2.2 Plant Cultivation
		2.3 Arabidopsis Transformation
		2.4 Selection of Transgenic Lines
		2.5 Confocal Laser Scanning Microscopy
	3 Methods
		3.1 Construction of Tagging Construct
		3.2 Cultivation of Arabidopsis
		3.3 Generation of Transgenic Lines
		3.4 Selection of Transgenic Lines
		3.5 Visualization
	4 Notes
	References
Chapter 15: Trichome Transcripts as Efficiency Control for Synthetic Biology and Molecular Farming
	1 Introduction
	2 Materials
		2.1 Plant Cultivation
		2.2 Trichome Harvest and Isolation
		2.3 Homogenization of Trichomes
		2.4 RNA Isolation with TRIsure
		2.5 RNA Cleanup (Optional)
		2.6 cDNA Synthesis of Targeted mRNA
	3 Methods
		3.1 Plant Cultivation
		3.2 Trichome Harvest and Isolation
		3.3 Homogenization of Trichomes
		3.4 RNA Isolation with TRIsure
		3.5 RNA Cleanup with RNA-Easy Columns (Optional)
		3.6 cDNA Synthesis of Targeted mRNA
	4 Notes
	References
Chapter 16: Generation of Stable, Light-Driven Co-cultures of Cyanobacteria with Heterotrophic Microbes
	1 Introduction
	2 Materials
		2.1 Cyanobacterial Strain
		2.2 Heterotrophic Strain
		2.3 Cell Culture Media and Prepared Solutions
			2.3.1 Preparation of Cell Growth Media
			2.3.2 Preparation of Additional Chemical Stocks
			2.3.3 Preparation of Sodium Alginate
			2.3.4 Preparation of Barium Chloride Curing Solution
	3 Methods
		3.1 Preparation of Cyanobacterial Culture
		3.2 Preparation of Heterotrophic Culture
		3.3 Encapsulation of S. elongatus CscB+
			3.3.1 Preparation of Cyanobacterial Cells
			3.3.2 Encapsulation of Cyanobacterial Cells
		3.4 Maturation of Beads
		3.5 Evaluation of Cell Concentration of Heterotrophs in Consortium
	4 Notes
	References
Index