DNA Cloning and Assembly, 1st ed. 2020 Methods and Protocols Methods in Molecular Biology Series, Vol. 2205

This volume provides a comprehensive collection of DNA assembly protocols that will prove useful for any researcher interested in molecular cloning, synthetic biology, or DNA manipulation. Chapters will guide readers through computational tools to design and track the construction of DNA assemblies, workflows that enable high-throughput assembly of DNA constructs, standardized toolkits and protocols for DNA assembly, and combinatorial solutions that enable the construction and optimization of entire metabolic pathways. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, DNA Cloning and Assembly: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Part I: In Silico Tools for DNA Design and Assembly

1. An Integrated Computer-Aided Design and Manufacturing Workflow for Synthetic Biology

Ernst Oberortner, Robert Evans, Xianwei Meng, Sangeeta Nath, Hector Plahar, Lisa Simirenko, Angela Tarver, Samuel Deutsch, Nathan J. Hillson, and  Jan-Fang Cheng

2. Combinatorial-Hierarchical DNA Library Design Using the TeselaGen DESIGN Module with j5

Michael J Fero, James K Craft, Trang Vu, and Nathan J Hillson

3. The AssemblX Toolkit for Reliable and User-friendly Multi-gene Assemblies

Part II: High Throughput Workflows

4. High-Throughput PCR for DNA Part Generation

David Reif

5. High-Throughput DNA Assembly Using Yeast Homologous Recombination

Kristy Ip, Ron Yadin, and Kevin W. George

6. Highly-Multiplexed, Semi-Automated Nextera Next Generation Sequencing (NGS) Library Preparation

William Christie, Ron Yadin, Kristy Ip, and Kevin W. George

7. Generation of MoClo Standard Parts Using Golden Gate Cloning

Ramona Grützner and Sylvestre Marillonnet

8. Assembly of Multigene Constructs using the Modular Cloning System MoClo

Sylvestre Marillonnet and Stefan Werner

9. Hierarchical Modular DNA Assembly using MetClo

Da Lin and Christopher A. O’Callaghan

10. PaperClip DNA Assembly: Reduce, Reuse, Recycle

Maryia Trubitsyna, Annegret Honsbein, Uma Jayachandran, Alistair Elfick, and Christopher E. French

11. Phytobricks: Manual and Automated Assembly of Constructs for Engineering Plants

Yao-Min Cai , Jose A. Carrasco Lopez, and Nicola J. Patron

12. Mobius Assembly

13. Design and Implementation of Multi-Protein Expression Constructs and Combinatorial Libraries using Start-Stop Assembly

George M. Taylor and John T. Heap

Marko Storch, Ari Dwijayanti,  Haris Mallick, and Matthew C. Haines,  and Geoff S. Baldwin

Marcos Valenzuela-Ortegaand Christopher E. French

Part IV: Combinatorial Methods For Pathway Optimization

Gita Naseriand Bernd Mueller-Roeber

17. SCRaMbLE-in: A Fast and Efficient Method to Diversify and Improve the Yields of Heterologous Pathways in Synthetic Yeast

Reem Swidah, Jamie Auxillos, Wei Liu, Sally Jones, Ting-Fung Chan, Junbiao Dai, and Yizhi Cai

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts