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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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Seamless assembly system M&C (meet-and-cut) Linear-TRAS.A Design of a seamless assembly system M&C (meet-and-cut) Linear-TRAS containing the unit vectors of C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. This system also includes the helper plasmids pM&CTRASHelperK, pM&CTRASHelperC and pM&CTRASHelperG for removing the kan, cat and gen markers respectively. I-CreI, I-SceI and I-PpoI in yellow rectangles indicate the homing endonuclease sites. Lx, La, Lb and Lc stand for the landing pad. I-CreI, I-SceI and I-PpoI in red triangle-ended rectangles indicate the genes encoding for the homing endonucleases. The alpha, beta and exo stand for the lambda red phage alpha, beta and exo genes. The recA stands for the E. coli recA gene. The assembly of two fragments K-DNA1-G and G-DNA2-C under the catalysis of pM&CTRASHelperG (B). Similar to the system described in Figure 5, PCR and counter-selection may be necessary for indentifying the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
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pone-0056854-g009: Seamless assembly system M&C (meet-and-cut) Linear-TRAS.A Design of a seamless assembly system M&C (meet-and-cut) Linear-TRAS containing the unit vectors of C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. This system also includes the helper plasmids pM&CTRASHelperK, pM&CTRASHelperC and pM&CTRASHelperG for removing the kan, cat and gen markers respectively. I-CreI, I-SceI and I-PpoI in yellow rectangles indicate the homing endonuclease sites. Lx, La, Lb and Lc stand for the landing pad. I-CreI, I-SceI and I-PpoI in red triangle-ended rectangles indicate the genes encoding for the homing endonucleases. The alpha, beta and exo stand for the lambda red phage alpha, beta and exo genes. The recA stands for the E. coli recA gene. The assembly of two fragments K-DNA1-G and G-DNA2-C under the catalysis of pM&CTRASHelperG (B). Similar to the system described in Figure 5, PCR and counter-selection may be necessary for indentifying the correct products to avoid the false positive case where two unit plasmids exist in the same cell.

Mentions: A final variation for TRAS, is a seamless model based on the MAGIC [74], Landing Pad [42] or recE [46] recombination systems. An example of a seamless linear Bi-Swap TRAS is shown for two DNA fragments in Figure 9, where the homing endonucleases expressed by the host strain are used to make site-specific double-strand breaks in the DNA. An alternative strategy is to express the required homing endonucleases on the assembly unit plasmids as each plasmid will be cut by the product of the other plasmid when both plasmids are present in the cell (Figure S1).


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

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

Seamless assembly system M&C (meet-and-cut) Linear-TRAS.A Design of a seamless assembly system M&C (meet-and-cut) Linear-TRAS containing the unit vectors of C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. This system also includes the helper plasmids pM&CTRASHelperK, pM&CTRASHelperC and pM&CTRASHelperG for removing the kan, cat and gen markers respectively. I-CreI, I-SceI and I-PpoI in yellow rectangles indicate the homing endonuclease sites. Lx, La, Lb and Lc stand for the landing pad. I-CreI, I-SceI and I-PpoI in red triangle-ended rectangles indicate the genes encoding for the homing endonucleases. The alpha, beta and exo stand for the lambda red phage alpha, beta and exo genes. The recA stands for the E. coli recA gene. The assembly of two fragments K-DNA1-G and G-DNA2-C under the catalysis of pM&CTRASHelperG (B). Similar to the system described in Figure 5, PCR and counter-selection may be necessary for indentifying the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585241&req=5

pone-0056854-g009: Seamless assembly system M&C (meet-and-cut) Linear-TRAS.A Design of a seamless assembly system M&C (meet-and-cut) Linear-TRAS containing the unit vectors of C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. This system also includes the helper plasmids pM&CTRASHelperK, pM&CTRASHelperC and pM&CTRASHelperG for removing the kan, cat and gen markers respectively. I-CreI, I-SceI and I-PpoI in yellow rectangles indicate the homing endonuclease sites. Lx, La, Lb and Lc stand for the landing pad. I-CreI, I-SceI and I-PpoI in red triangle-ended rectangles indicate the genes encoding for the homing endonucleases. The alpha, beta and exo stand for the lambda red phage alpha, beta and exo genes. The recA stands for the E. coli recA gene. The assembly of two fragments K-DNA1-G and G-DNA2-C under the catalysis of pM&CTRASHelperG (B). Similar to the system described in Figure 5, PCR and counter-selection may be necessary for indentifying the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
Mentions: A final variation for TRAS, is a seamless model based on the MAGIC [74], Landing Pad [42] or recE [46] recombination systems. An example of a seamless linear Bi-Swap TRAS is shown for two DNA fragments in Figure 9, where the homing endonucleases expressed by the host strain are used to make site-specific double-strand breaks in the DNA. An alternative strategy is to express the required homing endonucleases on the assembly unit plasmids as each plasmid will be cut by the product of the other plasmid when both plasmids are present in the cell (Figure 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