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A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

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Vectors for synthon recombination and transfer to expression plasmids.(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (1) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (2) to yield a composite product (3a), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (3b). Final assemblies (3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.
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pone-0037617-g002: Vectors for synthon recombination and transfer to expression plasmids.(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (1) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (2) to yield a composite product (3a), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (3b). Final assemblies (3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.

Mentions: The assembly procedure consists of three stages. First, entry vectors are digested with either of the two IIS endonucleases such that in each case the resulting entry synthon has a 4-base cohesive overhang exposed at one end while one of the orthogonal protecting groups is retained at the opposite end (Fig. 1B). Subsequently, complementary entry synthons are pooled and fused by ligation, yielding composite synthons that are idempotent to the entry synthons by restoration of the orthogonal protecting group configuration. At each assembly level, the synthons are digested in parallel with one of the IIS endonucleases essentially as in the first step. The procedure is reminiscent of split-and-pool assembly strategies frequently applied in combinatorial chemistry. Within this framework synthons can enter iterative or recursive assembly cycles until the required target construct is obtained (Fig. 1B). Since the assembly of orthogonally deprotected synthons is unidirectional, only a single product can be formed. Finally, the assembled product synthons can be ligated back into the empty entry vector, which may serve as a general repository vector (see Fig. 1A). Alternatively, the synthons can be directly cloned into a shuttle vector (pShuttle) for subsequent transfer into expression plasmids (Fig. 2A). In our laboratory we utilize a standardized set of expression vectors that uniformly contain 5′-BamHI and 3′-NotI restriction sites to facilitate rapid subcloning of constructs into vectors for different expression hosts and are equipped with a variety of tag- or signal peptide decorations. The pShuttle vector is a modified pCR8-TOPO vector carrying a stuffer sequence that is flanked on both sites by BsaI restriction sites, which create cohesive overhangs compatible with those from the deprotected synthons; and additional 5′-BamHI and 3′-NotI restriction sites compatible to our in-house library of expression vectors.


A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Vectors for synthon recombination and transfer to expression plasmids.(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (1) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (2) to yield a composite product (3a), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (3b). Final assemblies (3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369885&req=5

pone-0037617-g002: Vectors for synthon recombination and transfer to expression plasmids.(A) Deprotected synthons can be ligated into a BsaI-digested shuttle vector (pShuttle) that contains 5′-BamHI and 3′-NotI restriction sites compatible with our in-house collection of expression vectors. (B) For applications that require modular recombination or insertion of individual elements, synthons can be ligated into the modular assembly vectors pDA-N or pDA-C. pDA-N vectors that carry synthons encoding amino-terminal elements of protein constructs (1) can be combined with synthons encoding carboxyl-terminal elements from pDA-C vectors (2) to yield a composite product (3a), optionally leaving an additional entry point via BsaI restriction sites to insert additional synthons (3b). Final assemblies (3a and 4) contain 5′-BamHI and 3′-NotI restriction sites for transfer into expression vectors.
Mentions: The assembly procedure consists of three stages. First, entry vectors are digested with either of the two IIS endonucleases such that in each case the resulting entry synthon has a 4-base cohesive overhang exposed at one end while one of the orthogonal protecting groups is retained at the opposite end (Fig. 1B). Subsequently, complementary entry synthons are pooled and fused by ligation, yielding composite synthons that are idempotent to the entry synthons by restoration of the orthogonal protecting group configuration. At each assembly level, the synthons are digested in parallel with one of the IIS endonucleases essentially as in the first step. The procedure is reminiscent of split-and-pool assembly strategies frequently applied in combinatorial chemistry. Within this framework synthons can enter iterative or recursive assembly cycles until the required target construct is obtained (Fig. 1B). Since the assembly of orthogonally deprotected synthons is unidirectional, only a single product can be formed. Finally, the assembled product synthons can be ligated back into the empty entry vector, which may serve as a general repository vector (see Fig. 1A). Alternatively, the synthons can be directly cloned into a shuttle vector (pShuttle) for subsequent transfer into expression plasmids (Fig. 2A). In our laboratory we utilize a standardized set of expression vectors that uniformly contain 5′-BamHI and 3′-NotI restriction sites to facilitate rapid subcloning of constructs into vectors for different expression hosts and are equipped with a variety of tag- or signal peptide decorations. The pShuttle vector is a modified pCR8-TOPO vector carrying a stuffer sequence that is flanked on both sites by BsaI restriction sites, which create cohesive overhangs compatible with those from the deprotected synthons; and additional 5′-BamHI and 3′-NotI restriction sites compatible to our in-house library of expression vectors.

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

Show MeSH