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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

SRAS.Schematic of the Single-Selective-Marker Recombination Assembly System (SRAS) shown in Figure 1, as applied to the experimental assembly of Lux genes (A): Lux A, LuxB, LuxC, LuxD and LuxE. The screening procedures and results are identical to Figure 1. 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.
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pone-0056854-g003: SRAS.Schematic of the Single-Selective-Marker Recombination Assembly System (SRAS) shown in Figure 1, as applied to the experimental assembly of Lux genes (A): Lux A, LuxB, LuxC, LuxD and LuxE. The screening procedures and results are identical to Figure 1. 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.

Mentions: A series of short DNA components including the kanamycin-resistant operon and R6Kgamma from pKD13, the chloramphenicol-resistant operon (consisting of the cat gene and its promoter) derived from pKD3, the synthesized lambda attL1, attL2, attR1 and attR2, the HK022 attP and attR cloned from CRIM plasmid pAH70 and its integration product, the phi80 attL and attR cloned from the integration product of the CRIM plasmid pAH153, and a cloning site region from pBHR68 [48] were assembled into the two unit plasmids (pUnitP and pUnitR), the two extraction plasmids (pUnitExP and pUnitExR) and TARGET plasmid by recombinant DNA technology (Figure 3). The detailed procedures for the construction of these plasmids and sequences can be found in the corresponding Vexcutor file (vxt file; Construction of the Unit and Extraction Plasmids.vxt), which shows the individual steps involved in the construction of each of these plasmids and the sequences in silico (to view download the program from http://www.synthenome.com/ as described above and open the File S1).


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

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

SRAS.Schematic of the Single-Selective-Marker Recombination Assembly System (SRAS) shown in Figure 1, as applied to the experimental assembly of Lux genes (A): Lux A, LuxB, LuxC, LuxD and LuxE. The screening procedures and results are identical to Figure 1. 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585241&req=5

pone-0056854-g003: SRAS.Schematic of the Single-Selective-Marker Recombination Assembly System (SRAS) shown in Figure 1, as applied to the experimental assembly of Lux genes (A): Lux A, LuxB, LuxC, LuxD and LuxE. The screening procedures and results are identical to Figure 1. 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.
Mentions: A series of short DNA components including the kanamycin-resistant operon and R6Kgamma from pKD13, the chloramphenicol-resistant operon (consisting of the cat gene and its promoter) derived from pKD3, the synthesized lambda attL1, attL2, attR1 and attR2, the HK022 attP and attR cloned from CRIM plasmid pAH70 and its integration product, the phi80 attL and attR cloned from the integration product of the CRIM plasmid pAH153, and a cloning site region from pBHR68 [48] were assembled into the two unit plasmids (pUnitP and pUnitR), the two extraction plasmids (pUnitExP and pUnitExR) and TARGET plasmid by recombinant DNA technology (Figure 3). The detailed procedures for the construction of these plasmids and sequences can be found in the corresponding Vexcutor file (vxt file; Construction of the Unit and Extraction Plasmids.vxt), which shows the individual steps involved in the construction of each of these plasmids and the sequences in silico (to view download the program from http://www.synthenome.com/ as described above and open the File S1).

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