Limits...
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.

Show MeSH

Related in: MedlinePlus

DRAS.The design of the DRAS system is shown comprising chromosome host, pUnitExK, pUnitExC, pUnitK and pUnitC (A). The Swap/Extract assembly process is shown for two fragments DNA1 and DNA2 (B). The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. R6Kgamma is the R6Kγ conditional replication site when PI protein is available in the cell. Frt is the frt recombination site. Mutated lambda phage attachment sequences are designated as attL1, attL2, attR1 and attR2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585241&req=5

pone-0056854-g006: DRAS.The design of the DRAS system is shown comprising chromosome host, pUnitExK, pUnitExC, pUnitK and pUnitC (A). The Swap/Extract assembly process is shown for two fragments DNA1 and DNA2 (B). The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. R6Kgamma is the R6Kγ conditional replication site when PI protein is available in the cell. Frt is the frt recombination site. Mutated lambda phage attachment sequences are designated as attL1, attL2, attR1 and attR2.

Mentions: When the system contains more than one selective marker, the In/Out-Extract strategy can still be implemented with minor modifications. The In/Out steps can be simplified by the swap of the two selective markers. Extraction vectors are also required. A design of this method is shown in Figure 6. It is designated as a Swap-Extract strategy. Briefly, recombination at the reactive ends and topology breaking occur concurrently and successful recombinants are screened out by substitution of the antibiotic resistant unit. The choice of topology breakers will dramatically affect the complexity of the system. Since topology breakers need to remain reactive after recombination (to obey Rule 2), frt from yeast and loxP from the P1 Phage are better options than attachment sites that feature binary recombination. In addition, the chromosomal host here has the same exclusive selective effect as occurs for the SRAS. The experimental steps involved in the DRAS approach are provided in the three Vexcutor files provided in the Files S8, S9, S10; these include the steps required to construct the DRAS host strain, a library of the lux genes previously used to demonstrate the SRAS method in a form suitable for DRAS and steps required for an equivalent demonstration of recombination.


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

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

DRAS.The design of the DRAS system is shown comprising chromosome host, pUnitExK, pUnitExC, pUnitK and pUnitC (A). The Swap/Extract assembly process is shown for two fragments DNA1 and DNA2 (B). The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. R6Kgamma is the R6Kγ conditional replication site when PI protein is available in the cell. Frt is the frt recombination site. Mutated lambda phage attachment sequences are designated as attL1, attL2, attR1 and attR2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056854-g006: DRAS.The design of the DRAS system is shown comprising chromosome host, pUnitExK, pUnitExC, pUnitK and pUnitC (A). The Swap/Extract assembly process is shown for two fragments DNA1 and DNA2 (B). The chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. R6Kgamma is the R6Kγ conditional replication site when PI protein is available in the cell. Frt is the frt recombination site. Mutated lambda phage attachment sequences are designated as attL1, attL2, attR1 and attR2.
Mentions: When the system contains more than one selective marker, the In/Out-Extract strategy can still be implemented with minor modifications. The In/Out steps can be simplified by the swap of the two selective markers. Extraction vectors are also required. A design of this method is shown in Figure 6. It is designated as a Swap-Extract strategy. Briefly, recombination at the reactive ends and topology breaking occur concurrently and successful recombinants are screened out by substitution of the antibiotic resistant unit. The choice of topology breakers will dramatically affect the complexity of the system. Since topology breakers need to remain reactive after recombination (to obey Rule 2), frt from yeast and loxP from the P1 Phage are better options than attachment sites that feature binary recombination. In addition, the chromosomal host here has the same exclusive selective effect as occurs for the SRAS. The experimental steps involved in the DRAS approach are provided in the three Vexcutor files provided in the Files S8, S9, S10; these include the steps required to construct the DRAS host strain, a library of the lux genes previously used to demonstrate the SRAS method in a form suitable for DRAS and steps required for an equivalent demonstration of recombination.

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