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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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Efficient synthon assembly with split-and-pool reactions.(A) Equimolar amounts of BsaI or BsmBI deprotected 13FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (13FNIII)1 to (13FNIII)8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (13FNIII)8 construct.
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pone-0037617-g003: Efficient synthon assembly with split-and-pool reactions.(A) Equimolar amounts of BsaI or BsmBI deprotected 13FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (13FNIII)1 to (13FNIII)8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (13FNIII)8 construct.

Mentions: We tested the applicability of our approach during the construction of a series of expression cassettes harboring tandem arrays of homologous protein domains for single-molecule force spectroscopy. DNA synthons encoding Asn1813 to Thr1901 of a fibronectin type III (13FNIII) domain of human fibronectin [17] were prepared according to the design principles outlined in Fig. 1A. The flanking sequences were chosen such that digestion with BsaI produces a 5′-GGGG overhang capable of annealing with a 5′-CCCC overhang present on synthons restricted with BsmBI. The ligation site between synthons translates to a GlyGly sequence in the final protein. BsaI and BsmBI restricted synthons retain a protection group on one end with orthogonal 5′-AAAA overhangs. Entry synthons were processed during three recursive assembly cycles. Relatively short reaction times (10–15 min) at ligase concentrations of 1 unit sufficed to covalently link complementary synthons with excellent yield (Fig. 3A,B). We note, however, that we did not assess the efficiency of our approach with overhang sequences other than the cohesive GGGG/CCCC system exemplified in this report.


A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Efficient synthon assembly with split-and-pool reactions.(A) Equimolar amounts of BsaI or BsmBI deprotected 13FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (13FNIII)1 to (13FNIII)8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (13FNIII)8 construct.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369885&req=5

pone-0037617-g003: Efficient synthon assembly with split-and-pool reactions.(A) Equimolar amounts of BsaI or BsmBI deprotected 13FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of (13FNIII)1 to (13FNIII)8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the (13FNIII)8 construct.
Mentions: We tested the applicability of our approach during the construction of a series of expression cassettes harboring tandem arrays of homologous protein domains for single-molecule force spectroscopy. DNA synthons encoding Asn1813 to Thr1901 of a fibronectin type III (13FNIII) domain of human fibronectin [17] were prepared according to the design principles outlined in Fig. 1A. The flanking sequences were chosen such that digestion with BsaI produces a 5′-GGGG overhang capable of annealing with a 5′-CCCC overhang present on synthons restricted with BsmBI. The ligation site between synthons translates to a GlyGly sequence in the final protein. BsaI and BsmBI restricted synthons retain a protection group on one end with orthogonal 5′-AAAA overhangs. Entry synthons were processed during three recursive assembly cycles. Relatively short reaction times (10–15 min) at ligase concentrations of 1 unit sufficed to covalently link complementary synthons with excellent yield (Fig. 3A,B). We note, however, that we did not assess the efficiency of our approach with overhang sequences other than the cohesive GGGG/CCCC system exemplified in this report.

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
Related in: MedlinePlus