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Parallel in vivo DNA assembly by recombination: experimental demonstration and theoretical approaches.

Shi Z, Wedd AG, Gras SL - PLoS ONE (2013)

Bottom Line: Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests.A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here.The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Melbourne, Parkville, Victoria, Australia. shiz@student.unimelb.edu.au

ABSTRACT
The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.

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Related in: MedlinePlus

Assembly units and linear assembly.1F, 1R, 2F and 2R are the reactive ends. (A) The conformation of a typical fragment for assembly; (B) A typical assembly reaction between two linear fragments DNA1 and DNA2. The designation ‘-‘ represents the scar left after reaction of 1R and 2F.
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pone-0056854-g004: Assembly units and linear assembly.1F, 1R, 2F and 2R are the reactive ends. (A) The conformation of a typical fragment for assembly; (B) A typical assembly reaction between two linear fragments DNA1 and DNA2. The designation ‘-‘ represents the scar left after reaction of 1R and 2F.

Mentions: Effective DNA assembly requires specific and efficient reactions between any two specified ends of given DNA fragments. If unrelated sequences are to be excluded from assembly products, each fragment needs two tightly flanking reactive ends (recombination sites). A DNA fragment with two reactive ends is defined as an assembly unit. The assembly process for linear assembly units is simply the reaction of reactive ends. The internal sequence between the two reactive ends will not influence the assembly product. Two units in linear carriers can be assembled by one reaction between their reactive ends (see Figure 4). However, most of the stable replicable DNA molecules in the widely-used laboratory bacterial host E. coli are circular.


Parallel in vivo DNA assembly by recombination: experimental demonstration and theoretical approaches.

Shi Z, Wedd AG, Gras SL - PLoS ONE (2013)

Assembly units and linear assembly.1F, 1R, 2F and 2R are the reactive ends. (A) The conformation of a typical fragment for assembly; (B) A typical assembly reaction between two linear fragments DNA1 and DNA2. The designation ‘-‘ represents the scar left after reaction of 1R and 2F.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585241&req=5

pone-0056854-g004: Assembly units and linear assembly.1F, 1R, 2F and 2R are the reactive ends. (A) The conformation of a typical fragment for assembly; (B) A typical assembly reaction between two linear fragments DNA1 and DNA2. The designation ‘-‘ represents the scar left after reaction of 1R and 2F.
Mentions: Effective DNA assembly requires specific and efficient reactions between any two specified ends of given DNA fragments. If unrelated sequences are to be excluded from assembly products, each fragment needs two tightly flanking reactive ends (recombination sites). A DNA fragment with two reactive ends is defined as an assembly unit. The assembly process for linear assembly units is simply the reaction of reactive ends. The internal sequence between the two reactive ends will not influence the assembly product. Two units in linear carriers can be assembled by one reaction between their reactive ends (see Figure 4). However, most of the stable replicable DNA molecules in the widely-used laboratory bacterial host E. coli are circular.

Bottom Line: Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests.A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here.The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Melbourne, Parkville, Victoria, Australia. shiz@student.unimelb.edu.au

ABSTRACT
The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.

Show MeSH
Related in: MedlinePlus