Research Article pubs.acs.org/synthbio
EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors Andrea Martella,† Mantas Matjusaitis,‡ Jamie Auxillos,† Steven M. Pollard,*,‡ and Yizhi Cai*,† †
School of Biological Sciences, The University of Edinburgh, The King’s Buildings, Edinburgh EH9 3BF, U.K. MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh, EH16 4UU, U.K.
‡
ACS Synth. Biol. 2017.6:1380-1392. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/29/18. For personal use only.
S Supporting Information *
ABSTRACT: Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology. KEYWORDS: mammalian synthetic biology, mammalian expression vectors, DNA assembly, combinatorial assembly
M
dimerization domains also provides a wealth of possibilities for probing and perturbing protein function at the posttranslational level. Self-processing viral 2A peptides (p2A) or internal ribosomal entry site (IRES) elements have been widely deployed to enable production of multicistronic transcription units (TUs), thay is, multiple protein products generated from a mRNA molecule, and this is often useful for control of selectable markers, fluorescent reporters, or delivery of reprogramming factors.16,17 Expression cassettes can also be delivered as targeting vectors, for site-specific modification of endogenous genes: a critical tool of reverse genetics in mammalian development biology and studies of pluripotent stem cells. In short, a plethora of different options have emerged over the past decades for utilizing plasmid-based expression vectors across basic and applied biology. Despite the huge diversity and numbers of functional elements within plasmids as well as the wide-ranging applications, the vast majority of expression vectors are modular and they have shared genetic parts (i.e., DNA sequences conferring specific biological activity). A huge diversity of
ammalian expression vectors are a ubiquitous tool exploited across many areas of biological and biomedical research. They are used across diverse applications such as delivery of cDNAs, reporters, regulatory RNAs or other genetic elements into target cells; this can be achieved either transiently via plasmid-based or adenoviral vectors,1,2 or stably, through integration to the target cell genome via transposon-based vectors, vectors for homologous recombination-based gene targeting or episomal vectors.2−7 This has transformed our ability to study fundamental processes governing gene and cellular regulation across many mammalian species. A similarly profound impact has been made across biotechnology such as the production of recombinant proteins, engineering of stem cells, or use in gene therapy.8−10 Expression vectors can be designed to incorporate distinct transcriptional regulatory elements, such as constitutive or inducible promoters (natural or synthetic) that drive either distinct levels or lineage-specific patterns of gene expression.11 Additionally post-transcriptional regulatory elements typically placed in the 5′ or 3′ untranslated regions (UTR) can influence the mRNA stability,12−14 and different transcriptional terminators may also have an impact on gene expression.15 Reengineering natural proteins with upstream or downstream peptides, protein degradation tags, linker peptides, or © 2017 American Chemical Society
Received: January 16, 2017 Published: April 18, 2017 1380
DOI: 10.1021/acssynbio.7b00016 ACS Synth. Biol. 2017, 6, 1380−1392
Research Article
ACS Synthetic Biology
Figure 1. EMMA platform for the design of mammalian expression vectors. (A) Platform used to design the expression vectors. Positions in the platform are labeled with numbers (1−25). The 4 bp fusion sites are labeled with letters (A−Z). Functional categories are represented by standardized schematic glyphs according to the Synthetic Biology Open Language (SBOL) or assigned by the authors when not available in the SBOL standards. One or more categories have been assigned to each position in the platform. The red bars on the top of the platform represent the positions covered by the assembly connector; the letters on both sides of the red bars indicate the flanking fusion sites. (B) Key for the schematic glyphs representing the functional categories. TU, transcription unit; ITR, inverted terminal repeat; LTR, long terminal repeat; CDS, coding sequence; IRES, internal ribosome entry site; UTR, untranslated region.
assembly,21 and Golden Gate (GG) assembly.22 These methods have the key feature of enabling efficient assembly of multiple reusable DNA parts in a single reaction and have paved the way for the modular cloning, an engineering-inspired strategy using standardized parts and robust and reliable assembly rules. Modular Cloning (MoClo) and GoldenBraid23−25 are modular GG-based cloning techniques that have been reported to provide efficient directional assembly of transcription units (TUs) using a library of readily made standardized parts. These systems allow parts to be exchanged and assembled cheaply, easily, and potentially in an automatable fashion. MoClo has been recently applied in mammalian systems,26 and is useful for hierarchical multistep assembly of multigene genetic circuits. However, it does not fully address the requirements for generation of functionally diverse mammalian expression vectors with a large numbers of parts. GMAP is an alternative method based on Gibson assembly,27 and can be used for generation of mammalian expression vectors, but is limited in flexibility and numbers of parts (only five); however, typical mammalian expression vectors require ∼10−20 functionally distinct parts. Furthermore, Gibson assembly relies on overlapping flanks and in vitro recombination, which limits both the possibilities for assembly of short DNA fragments and the reuse of these parts. Many components of an expression unit can be
