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.
Highlights
The design and construction of DNA sequences by assembly of modular DNA fragments lies at the core of protein engineering
General Assembly Strategy A restriction and ligation-based approach to DNA assembly is introduced that follows the general concept of protecting groupbased chemical synthesis strategies
We have demonstrated that the application of IIS restriction endonucleases can be readily combined with a simple protecting group concept to create a versatile framework for the rapid assembly of modular DNA building blocks into functional gene constructs
Summary
The design and construction of DNA sequences by assembly of modular DNA fragments lies at the core of protein engineering. The advent of synthetic biology with associated technological advances in manufacturing of DNA sequences has recently matured de novo synthesis of custom genes into an important resource In spite of these advances, there remains a continuous demand for robust and cost-effective alternatives, adaptable by individual laboratories, to aid downstream processing of DNA sequences. Chemical synthesis strategies frequently employ transient protecting groups that allow modular building blocks (synthons) to be assembled at specific reactive sites in a defined synthetic sequence. This concept is supported by the ability to selectively remove these protecting groups with a set of mutually exclusive (orthogonal) reaction conditions. We here validate applicability of these principles to the assembly of expression cassettes encoding tandem arrays of protein domains for single-molecule force spectroscopy
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