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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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Linear Bi-Swap TRAS.The schematic design of linear Bi-Swap TRAS containing the unit vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A). The yellow round hairpin ends represent N15 plasmids. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. The assembly process for five DNA fragments DNA1, DNA2, DNA3, DNA4 and DNA5 (B). Because this is a linear plasmid system, theoretically no fusion intermediate could form after recombination. PCR and counter-selection may be necessary to indentify the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
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pone-0056854-g008: Linear Bi-Swap TRAS.The schematic design of linear Bi-Swap TRAS containing the unit vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A). The yellow round hairpin ends represent N15 plasmids. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. The assembly process for five DNA fragments DNA1, DNA2, DNA3, DNA4 and DNA5 (B). Because this is a linear plasmid system, theoretically no fusion intermediate could form after recombination. PCR and counter-selection may be necessary to indentify the correct products to avoid the false positive case where two unit plasmids exist in the same cell.

Mentions: In E. coli and many other prokaryotes, there are also linear plasmids such as the N15 phage plasmids [73]. In the case of linear plasmids or genomes, the linear ends are ideal topology breakers because no recombination at the topology breakers is required (although telomerase can be considered as a kind of recombinase). Therefore, it is possible to use a single recombination strategy to finish the whole assembly process. Figure 8 shows a possible design for a linear Bi-Swap strategy where five DNA fragments are assembled in order. The steps involved in the linear DRAS and linear TRAS strategies are presented in a Vexcutor file in the File S12. Two more advanced Bi-Swap BioBrick examples are also presented in Figure S2 and S3. These approaches are an extension on those presented in Figure 7 and 8 that employ recombination sites as the reactive ends that drive assembly.


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

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

Linear Bi-Swap TRAS.The schematic design of linear Bi-Swap TRAS containing the unit vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A). The yellow round hairpin ends represent N15 plasmids. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. The assembly process for five DNA fragments DNA1, DNA2, DNA3, DNA4 and DNA5 (B). Because this is a linear plasmid system, theoretically no fusion intermediate could form after recombination. PCR and counter-selection may be necessary to indentify the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056854-g008: Linear Bi-Swap TRAS.The schematic design of linear Bi-Swap TRAS containing the unit vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A). The yellow round hairpin ends represent N15 plasmids. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. The assembly process for five DNA fragments DNA1, DNA2, DNA3, DNA4 and DNA5 (B). Because this is a linear plasmid system, theoretically no fusion intermediate could form after recombination. PCR and counter-selection may be necessary to indentify the correct products to avoid the false positive case where two unit plasmids exist in the same cell.
Mentions: In E. coli and many other prokaryotes, there are also linear plasmids such as the N15 phage plasmids [73]. In the case of linear plasmids or genomes, the linear ends are ideal topology breakers because no recombination at the topology breakers is required (although telomerase can be considered as a kind of recombinase). Therefore, it is possible to use a single recombination strategy to finish the whole assembly process. Figure 8 shows a possible design for a linear Bi-Swap strategy where five DNA fragments are assembled in order. The steps involved in the linear DRAS and linear TRAS strategies are presented in a Vexcutor file in the File S12. Two more advanced Bi-Swap BioBrick examples are also presented in Figure S2 and S3. These approaches are an extension on those presented in Figure 7 and 8 that employ recombination sites as the reactive ends that drive assembly.

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