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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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Bi-Swap TRAS.The design of Bi-Swap TRAS containing the vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where CF, CR, GR, GF, KR and KF are verification primer sites. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. In order to screen a recombination product with a new combination of antibiotic resistance, at least three antibiotic resistance markers must be used. Since the DNA sequence is directional and two antibiotic resistance markers must be different to perform screening, the total number of Unit plasmids is 6 (3×2). In this case, an assembly Unit vector has one Unit vector that can swap and extend its left arm or one Unit vector that can swap and extend its right arm. For example, if C-DNA1-G is the beginning unit, K-DNA2-C can be used to extend the left arm of C-DNA1-G and G-DNA2-K can be used to extend the right arm of C-DNA1-G. The assembly process of the fragments DNA1 and DNA2 (B). G-DNA2-K is used to swap and extend the right arm of C-DNA1-G. Because this is a circular plasmid system, fusion intermediates could form after recombination (but will contain all three antibiotic resistant markers). Therefore, PCR and counter-selection are considered necessary to indentify the correct products.
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pone-0056854-g007: Bi-Swap TRAS.The design of Bi-Swap TRAS containing the vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where CF, CR, GR, GF, KR and KF are verification primer sites. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. In order to screen a recombination product with a new combination of antibiotic resistance, at least three antibiotic resistance markers must be used. Since the DNA sequence is directional and two antibiotic resistance markers must be different to perform screening, the total number of Unit plasmids is 6 (3×2). In this case, an assembly Unit vector has one Unit vector that can swap and extend its left arm or one Unit vector that can swap and extend its right arm. For example, if C-DNA1-G is the beginning unit, K-DNA2-C can be used to extend the left arm of C-DNA1-G and G-DNA2-K can be used to extend the right arm of C-DNA1-G. The assembly process of the fragments DNA1 and DNA2 (B). G-DNA2-K is used to swap and extend the right arm of C-DNA1-G. Because this is a circular plasmid system, fusion intermediates could form after recombination (but will contain all three antibiotic resistant markers). Therefore, PCR and counter-selection are considered necessary to indentify the correct products.

Mentions: When three or more selective markers are available, both the In/Out-Extract and Swap/Extract strategies can be implemented. Moreover, the selective marker swap strategy can be applied between unit plasmids so that integration into the host is not necessary and the extraction step is not required. This is designated a bidirectional swap (Bi-Swap) strategy. A typical design employing the Bi-Swap strategy for the directional assembly of two DNA fragments is shown in Figure 7. The steps involved in the application of TRAS are shown in the Vexcutor file in the File S11.


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

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

Bi-Swap TRAS.The design of Bi-Swap TRAS containing the vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where CF, CR, GR, GF, KR and KF are verification primer sites. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. In order to screen a recombination product with a new combination of antibiotic resistance, at least three antibiotic resistance markers must be used. Since the DNA sequence is directional and two antibiotic resistance markers must be different to perform screening, the total number of Unit plasmids is 6 (3×2). In this case, an assembly Unit vector has one Unit vector that can swap and extend its left arm or one Unit vector that can swap and extend its right arm. For example, if C-DNA1-G is the beginning unit, K-DNA2-C can be used to extend the left arm of C-DNA1-G and G-DNA2-K can be used to extend the right arm of C-DNA1-G. The assembly process of the fragments DNA1 and DNA2 (B). G-DNA2-K is used to swap and extend the right arm of C-DNA1-G. Because this is a circular plasmid system, fusion intermediates could form after recombination (but will contain all three antibiotic resistant markers). Therefore, PCR and counter-selection are considered necessary to indentify the correct products.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056854-g007: Bi-Swap TRAS.The design of Bi-Swap TRAS containing the vectors C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K (A), where CF, CR, GR, GF, KR and KF are verification primer sites. The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. In order to screen a recombination product with a new combination of antibiotic resistance, at least three antibiotic resistance markers must be used. Since the DNA sequence is directional and two antibiotic resistance markers must be different to perform screening, the total number of Unit plasmids is 6 (3×2). In this case, an assembly Unit vector has one Unit vector that can swap and extend its left arm or one Unit vector that can swap and extend its right arm. For example, if C-DNA1-G is the beginning unit, K-DNA2-C can be used to extend the left arm of C-DNA1-G and G-DNA2-K can be used to extend the right arm of C-DNA1-G. The assembly process of the fragments DNA1 and DNA2 (B). G-DNA2-K is used to swap and extend the right arm of C-DNA1-G. Because this is a circular plasmid system, fusion intermediates could form after recombination (but will contain all three antibiotic resistant markers). Therefore, PCR and counter-selection are considered necessary to indentify the correct products.
Mentions: When three or more selective markers are available, both the In/Out-Extract and Swap/Extract strategies can be implemented. Moreover, the selective marker swap strategy can be applied between unit plasmids so that integration into the host is not necessary and the extraction step is not required. This is designated a bidirectional swap (Bi-Swap) strategy. A typical design employing the Bi-Swap strategy for the directional assembly of two DNA fragments is shown in Figure 7. The steps involved in the application of TRAS are shown in the Vexcutor file in the File S11.

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