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This volume provides an overview of the latest protocols that cover the full range of in silico, in vitro, and in vivo approaches to help researchers plan, design, and perform Golden Gate cloning experiments. The chapters in this book cover topics such as Golden Gate cloning of standardized parts; modular DNA construct design for high-throughput Golden Gate Assembly; Golden Gate cloning of Synthetic CRISPR RNA spacer sequences; Golden Gate cloning in Actinobacteria; and YeastFab cloning of toxic genes and protein expression optimization in yeast. 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 thorough, Golden Gate Cloning: Methods and Protocols is a valuable resource for all researchers interested in learning more about this important and developing field.
Chapter 3 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
List of contents
Golden Gate Cloning of MoClo Standard Parts.- Assembly of Multigene Constructs using the Modular Cloning System MoClo.- Selection of Fusion-Site Overhang Sets for High Fidelity and High Complexity Golden Gate Assembly.- Modular DNA Construct Design for High-Throughput Golden Gate Assembly.- Using ApE for In Silico Golden Gate Cloning.- Standardized Golden Gate Assembly Metadata Representation using SBOL.- Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.- Biofoundry-Assisted Golden Gate Cloning with AssemblyTron.- Automation and Miniaturization of Golden Gate DNA Assembly Reactions using Acoustic Dispensers.- Validation of Golden Gate Assemblies using Highly Multiplexed Nanopore Amplicon Sequencing.- Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.- Construction of Small Genes with Repetitive Elements using Oligo Extension and Golden Gate Assembly.- Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.- Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids using Golden Gate Cloning.- Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.- Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.- Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.- Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.- Golden Gate Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.- Protocol for NT-CRISPR, A Method for Efficient Genome Engineering in Vibrio natriegens.- Golden Gate Cloning in Actinobacteria: Opportunities and Challenges.- Golden Gate Assembly of Transcriptional Unit Libraries into a Rearrangeable Gene Cluster.- Golden Gate Cloning for Efficient Biosynthesis of Lycopene in Synthetic Yeast.- YeastFab Cloning of Toxic Genes and Protein Expression Optimization in Yeast.- Golden Gate Cloning for the Standardized Assembly of Gene Elements with Modular Cloning in Chlamydomonas.- Assembling DNA Plasmids with the Multi-Kingdom (MK) Cloning System.- Advancements in Golden Gate Cloning: A Comprehensive Review.
