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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 process details of SRAS.The schematic design of the Single-Selective-Marker Recombination Assembly System (SRAS) is shown here (A), consisting of two unit vectors pUnitR, pUnitP, the excision vectors pUnitExR and pUnitExP and the E.coli host chromosome. One method of I/O assembly is shown here for the assembly of two DNA fragments DNA1 and DNA 2 (B): integration occurs via reactive ends first and excision via topology breakers. A second method of I/O assembly is shown here for the assembly of two DNA fragments DNA 1 and DNA2 (C): integration occurs via topology breakers first and excision via reactive ends. The screening procedures and results are identical to B part of this figure. attLPhi80 and attPPhi80 stand for the Phi80 phage attL and attR, respectively. attLHK022, attRHK022, attBHK022 and attPHK022 stand for the HK022 phage attL, attR, attB and attP respectively. The chloroamphenicol and kanamycin resistance genes are designated cat and kan respectively. The cat gene is not employed in the schematic above but could be used as a second single selective marker in parallel rounds of DNA assembly.
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pone-0056854-g001: Assembly process details of SRAS.The schematic design of the Single-Selective-Marker Recombination Assembly System (SRAS) is shown here (A), consisting of two unit vectors pUnitR, pUnitP, the excision vectors pUnitExR and pUnitExP and the E.coli host chromosome. One method of I/O assembly is shown here for the assembly of two DNA fragments DNA1 and DNA 2 (B): integration occurs via reactive ends first and excision via topology breakers. A second method of I/O assembly is shown here for the assembly of two DNA fragments DNA 1 and DNA2 (C): integration occurs via topology breakers first and excision via reactive ends. The screening procedures and results are identical to B part of this figure. attLPhi80 and attPPhi80 stand for the Phi80 phage attL and attR, respectively. attLHK022, attRHK022, attBHK022 and attPHK022 stand for the HK022 phage attL, attR, attB and attP respectively. The chloroamphenicol and kanamycin resistance genes are designated cat and kan respectively. The cat gene is not employed in the schematic above but could be used as a second single selective marker in parallel rounds of DNA assembly.

Mentions: A schematic diagram of the method tested experimentally using the Single-Selective-Marker Recombination Assembly System (SRAS) for parallel DNA assembly developed here is given in Figure 1. The next section describes the preparation of the experimental tools required for SRAS, followed by a description of methods involved in the experimental demonstration of SRAS.


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

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

Assembly process details of SRAS.The schematic design of the Single-Selective-Marker Recombination Assembly System (SRAS) is shown here (A), consisting of two unit vectors pUnitR, pUnitP, the excision vectors pUnitExR and pUnitExP and the E.coli host chromosome. One method of I/O assembly is shown here for the assembly of two DNA fragments DNA1 and DNA 2 (B): integration occurs via reactive ends first and excision via topology breakers. A second method of I/O assembly is shown here for the assembly of two DNA fragments DNA 1 and DNA2 (C): integration occurs via topology breakers first and excision via reactive ends. The screening procedures and results are identical to B part of this figure. attLPhi80 and attPPhi80 stand for the Phi80 phage attL and attR, respectively. attLHK022, attRHK022, attBHK022 and attPHK022 stand for the HK022 phage attL, attR, attB and attP respectively. The chloroamphenicol and kanamycin resistance genes are designated cat and kan respectively. The cat gene is not employed in the schematic above but could be used as a second single selective marker in parallel rounds of DNA assembly.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585241&req=5

pone-0056854-g001: Assembly process details of SRAS.The schematic design of the Single-Selective-Marker Recombination Assembly System (SRAS) is shown here (A), consisting of two unit vectors pUnitR, pUnitP, the excision vectors pUnitExR and pUnitExP and the E.coli host chromosome. One method of I/O assembly is shown here for the assembly of two DNA fragments DNA1 and DNA 2 (B): integration occurs via reactive ends first and excision via topology breakers. A second method of I/O assembly is shown here for the assembly of two DNA fragments DNA 1 and DNA2 (C): integration occurs via topology breakers first and excision via reactive ends. The screening procedures and results are identical to B part of this figure. attLPhi80 and attPPhi80 stand for the Phi80 phage attL and attR, respectively. attLHK022, attRHK022, attBHK022 and attPHK022 stand for the HK022 phage attL, attR, attB and attP respectively. The chloroamphenicol and kanamycin resistance genes are designated cat and kan respectively. The cat gene is not employed in the schematic above but could be used as a second single selective marker in parallel rounds of DNA assembly.
Mentions: A schematic diagram of the method tested experimentally using the Single-Selective-Marker Recombination Assembly System (SRAS) for parallel DNA assembly developed here is given in Figure 1. The next section describes the preparation of the experimental tools required for SRAS, followed by a description of methods involved in the experimental demonstration of SRAS.

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