Synthetic DNA Assembly and Genome Editing

Methods of plant breeding

1. Traditional

2. Genetic Engineering

Synthetic Biology

Application of engineering principles to the fundamental components of biology.

Aims for creating useful functions in diverse areas:

• Therapeutics

• Biosensors

• Bioenergy

• Bioremediation

• Chemicals and materials production

Key enabling technologies of synthetic biology

1. Computational modelling and quantitative measurement: modelling of the design to system system performance prior to fabrication.

2. DNA sequencing: The sequencing of entire genomes of numerous organisms has provided:

   • information regarding the framework within which synthetic biologists seek to construct functional devices.

   • genetic building blocks

3. DNA synthesis:

   • Chemical synthesis of fragments up to 1000 bp.

   • Molecular biology tools to assemble final synthesis constructs.

Booting life

Full genome synthesis of micro organisms

DNA assembly methods

1. DNA synthesis

2. By restriction/ligation

   • Traditional cloning in multiple cloning sites

   • BioBrick approach

   • Golden Gate shuffling

3. By homologous recombination

   • Sequence and ligation independent cloning (SLIC)

   • Gibson assembly

   • Circular polymerase extension cloning (CPEC)

   • In-fusion cloning

   • DNA shuffling

DNA synthesis as a DNA assembly method

Enzymatic methods for gene synthesis

1. Ligation-based assembly: usually involves two steps:

2. Two-step PCR-driven assembly

3. One-step gene assembly reaction

Explain the traditional cloning in multiple cloning sites approach as a DNA assembly method by restriction/ligation

Explain the BioBrick approach as a DNA assembly method by restriction/ligation

BioBricks are DNA sequences standardized according to a specific standard (BioBrick standard, BglBrick standard…), facilitating automatization and re-use.

BglBrick standard

BioBrick limitations and obstacles

• Scars: no control over the existence and sequence of intervening scars.

  • This is problematic when located in the coding sequence.

  • It can affect mRNA secondary structures.

• Iterative process: because only two blocks can be fused at a time, multiple DNA assembly requires an iterative process.

• Difficulty of creating a combinatorial library: especially when more than 2 BioBricks are combined.

Type IIS restriction enzyme

Explain the Golden Gate shuffling approach as a DNA assembly method by restriction/ligation

Golden Gate shuffling relies on two unique properties of type IIS restriction enzymes.

Limitations and obstacles of Golden Gate shuffling

Sequence and Ligation Independent Cloning (SLIC)

Gibson Assembly

Circular Polymerase Extension Cloning (CPEC)

Clonetech In-Fusion Cloning kit

Zinc Finger Nucleases (ZFN)

Fusion of:

• DNA binding domain

• DNA cleavage domain

Talen

Transcription Activator-Like Effector Nucleases

Fusion of:

• DNA binding domain

• DNA cleavage domain

CRISPR-Cas