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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

The experimental materials constructed for the demonstration of the Single-Selective-Marker Recombination Assembly System (SRAS).(A) The pUnitP and pUnitR unit vectors for constructing fragment libraries. (B) The pUnitExP and pUnitExR extraction vectors. (C) The assembly and helper Vectors that integrate the library vectors pUnitP and pUnitR into the host cell chromosome. (D) The extraction helper vectors. The helper vector contains a pir gene that allows the replication of the extracted vectors. (E) The host strain containing the TARGET sequence for SRAS.
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pone-0056854-g002: The experimental materials constructed for the demonstration of the Single-Selective-Marker Recombination Assembly System (SRAS).(A) The pUnitP and pUnitR unit vectors for constructing fragment libraries. (B) The pUnitExP and pUnitExR extraction vectors. (C) The assembly and helper Vectors that integrate the library vectors pUnitP and pUnitR into the host cell chromosome. (D) The extraction helper vectors. The helper vector contains a pir gene that allows the replication of the extracted vectors. (E) The host strain containing the TARGET sequence for SRAS.

Mentions: A number of tools are required for the experimental demonstration of SRAS. These include the two unit plasmids (pUnitP and pUnitR), the two extraction plasmids (pUnitExP and pUnitExR), the TARGET plasmid within the host Escherichia coli (E. coli) and helper plasmids that assist with integration and excision of DNA. These tools are shown in Figure 2. The construction of these tools, integration of the TARGET plasmid and modification of unit plasmids to incorporate the T7 promoter and terminator sequences is described below.


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

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

The experimental materials constructed for the demonstration of the Single-Selective-Marker Recombination Assembly System (SRAS).(A) The pUnitP and pUnitR unit vectors for constructing fragment libraries. (B) The pUnitExP and pUnitExR extraction vectors. (C) The assembly and helper Vectors that integrate the library vectors pUnitP and pUnitR into the host cell chromosome. (D) The extraction helper vectors. The helper vector contains a pir gene that allows the replication of the extracted vectors. (E) The host strain containing the TARGET sequence for SRAS.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056854-g002: The experimental materials constructed for the demonstration of the Single-Selective-Marker Recombination Assembly System (SRAS).(A) The pUnitP and pUnitR unit vectors for constructing fragment libraries. (B) The pUnitExP and pUnitExR extraction vectors. (C) The assembly and helper Vectors that integrate the library vectors pUnitP and pUnitR into the host cell chromosome. (D) The extraction helper vectors. The helper vector contains a pir gene that allows the replication of the extracted vectors. (E) The host strain containing the TARGET sequence for SRAS.
Mentions: A number of tools are required for the experimental demonstration of SRAS. These include the two unit plasmids (pUnitP and pUnitR), the two extraction plasmids (pUnitExP and pUnitExR), the TARGET plasmid within the host Escherichia coli (E. coli) and helper plasmids that assist with integration and excision of DNA. These tools are shown in Figure 2. The construction of these tools, integration of the TARGET plasmid and modification of unit plasmids to incorporate the T7 promoter and terminator sequences is described below.

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