Combinatorial DNA assembly using Golden Gate cloning.

A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. We have previously reported the development of Golden Gate cloning, a method that allows parallel assembly of multiple DNA fragments in a one-tube reaction. Golden Gate cloning can be used for different levels of construct assembly: from gene fragments to complete gene coding sequences, from basic genetic elements to full transcription units, and finally from transcription units to multigene constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying all DNA fragments by PCR to add flanking restriction sites, (3) cloning the amplified fragments in intermediate constructs, and (4) assembling all or selected sets of intermediate constructs in a compatible recipient vector using a one-pot restriction-ligation.

[1]  Quinn Lu,et al.  Seamless cloning and gene fusion , 2005, Trends in Biotechnology.

[2]  T. Nagai,et al.  A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme. , 2008, Journal of biotechnology.

[3]  Y. Berlin.  DNA splicing by directed ligation (SDL). , 1999, Current issues in molecular biology.

[4]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[5]  A general one‐step method for the cloning of PCR products , 2005, Biotechnology and applied biochemistry.

[6]  A. Granell,et al.  GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules , 2011, PloS one.

[7]  S. Elledge,et al.  Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC , 2007, Nature Methods.

[8]  Ernst Weber,et al.  A Modular Cloning System for Standardized Assembly of Multigene Constructs , 2011, PloS one.

[9]  Method of artificial DNA splicing by directed ligation (SDL). , 1991, Nucleic acids research.

[10]  Robert M. Horton,et al.  Gene splicing by overlap extension. , 1990 .

[11]  W. P. Stemmer,et al.  Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. , 1992, BioTechniques.

[12]  H. Dinse,et al.  Human Umbilical Cord Blood Cells Restore Brain Damage Induced Changes in Rat Somatosensory Cortex , 2011, PloS one.

[13]  Sylvestre Marillonnet,et al.  Assembly of Designer TAL Effectors by Golden Gate Cloning , 2011, PloS one.

[14]  Carola Engler,et al.  Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes , 2009, PloS one.

[15]  Z. G. Liu,et al.  An efficient method for blunt-end ligation of PCR products. , 1992, BioTechniques.

[16]  W Szybalski,et al.  Class-IIS restriction enzymes--a review. , 1991, Gene.

[17]  Tom Ellis,et al.  DNA assembly for synthetic biology: from parts to pathways and beyond. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[18]  Sylvestre Marillonnet,et al.  Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system , 2012 .

[19]  T. Lahaye,et al.  Assembly of custom TALE-type DNA binding domains by modular cloning , 2011, Nucleic acids research.

[20]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[21]  Jens Boch,et al.  Transcriptional Activators of Human Genes with Programmable DNA-Specificity , 2011, PloS one.

[22]  S. Ho,et al.  Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. , 2013, BioTechniques.

[23]  J Craig Venter,et al.  One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome , 2008, Proceedings of the National Academy of Sciences.

[24]  Neville E Sanjana,et al.  A transcription activator-like effector toolbox for genome engineering , 2012, Nature Protocols.

[25]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.