Abstract
Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.
Highlights
Synthetic biology is facilitated by DNA assembly systems which allow rapid generation of new constructs [1]
It was decided: that vectors would be based on the Standard European Vector Architecture (SEVA) system, which offers wide flexibility and applicability to different bacterial species; that level 0 parts should be based on the PhytoBrick standard, already accepted by iGEM and used in multiple other Golden Gate-based kits; that BioBrick compatibility would be included, for backwards compatibility with constructs in the Registry of Standard Biological Parts; and that the main cloning site in all level 1 and level 2 vectors would be flanked by upstream and downstream secondary sites, which would allow both level 1 and level 2 assembly directly into these sites
The JUMP ‘cargo’ (Figure 2B), consisting of upstream cloning site flanked by AarI sites, BioBrick prefix, main modular cloning site consisting of marker gene sfGFP flanked by BsaI and BsmBI sites, BioBrick suffix and downstream cloning site flanked by BbsI sites, was introduced
Summary
Synthetic biology is facilitated by DNA assembly systems which allow rapid generation of new constructs [1]. Synthetic biology researchers have to reiterate the design–build–test cycle several times until the new system is optimized or tuned and, DNA assembly standards that are robust, automatable and accept reusable DNA parts are still necessary [5]. While Gibson assembly [6] and other overlap-based methodologies (as reviewed by Casini et al [7]) are very efficient in assembling multiple DNA parts, standards based on Golden Gate cloning are the best fit for automation and part reusability [7]. In Golden Gate cloning [8], DNA parts are flanked by sites recognized by type IIS restriction enzymes, which cut outside the recognized site, leaving user-specified overhangs (or fusion sites). Different hierarchical standards based on Golden Gate have been published [10,11,12,13,14,15,16,17,18,19], often generically referred to as modular
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