Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

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.

Keywords: DNA assembly; Golden Gate cloning; SEVA; modular cloning; synthetic biology.

Figure 1.

Modular cloning and general design of Joint Universal Modular Plasmids. (A) Basic parts are contained in level 0 vectors, which are assembled to form a single Transcription Unit (TU) in a level 1 vector (either 1A, 1B, 1C or 1D). To simplify cloning and screening, the destination vector contains a different selection marker than insert-donor plasmids and a reporter gene replaced by the assembled TU. Using a second type IIS restriction enzyme and selection marker, multiple level 1 assemblies (1A, 1B, 1C and 1D) can be combined in a level 2 vector (2A, 2B, 2C or 2D). In JUMP, level 1 plasmids can be used as level 3 assembly destination vectors. (B) The design of JUMP vectors combines modular cloning in the main site (as shown in A), compatibility with BioBricks and SEVA, and two orthogonal secondary modular cloning sites to introduce new features to any vector.

Figure 2.

JUMP design and secondary sites. (A) In Standard European Vector Architecture (SEVA) plasmids, three common short DNA sequences (black) flank three variable regions (colored). Variable regions are the OriV (origin of replication), AbR (antibiotic selection marker) and ‘cargo’ (any expression cassette). The invariable regions are two transcription terminators flanking the cargo (T1 and T0) and origin of conjugation (oriT). Invariable regions also contain rare cutting sites, forbidden in the sequence of variable regions. (B) JUMP is designed as a special cargo of SEVA vectors to allow compatibility with future OriV's and AbR's of the collection. The cargo contains the upstream modular site (with outward-facing AarI sites); BioBrick prefix (XbaI, EcoRI); main modular site (a screening reporter gene flanked by outwards-facing BsaI and inwards-facing BsmBI sites for level 1, and vice versa for level 2); BioBrick suffix (SpeI, PstI); and downstream modular site (with outwards-facing BbsI sites). SEVA's canonical SpeI site was removed to allow BioBrick compatibility. (C) Building constructs to test similar genes (G¹ to Gⁿ) as sequences of interest (SOI) that depend on common auxiliary factors (Aux.) with conventional modular cloning might require multiple assembly steps per SOI: the SOI is first assembled by itself and then combined with the auxiliary elements. (D) Introduction of the auxiliary factors in vector chassis using orthogonal use of secondary sites reduces number of assembly steps to combine the SOI with the auxiliary factor. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green).

Figure 3.

JUMP nomenclature and backbones. (A) JUMP follows SEVA nomenclature for origins of replication (OriV) and selection marker (AbR), while cargo nomenclature is replaced by JUMP's main site module index. (B) Antibiotic selection markers used in JUMP vectors. (C) Origins of replication in JUMP vectors. (D) Default main site modules and their donor fusion sites. (E) OD-normalized fluorescence of sfGFP cloning reporter in available combinations of OriV-AbR in the distributed JUMP toolkit. Error bars indicate standard deviation, n = 3.

Figure 4.

Use of secondary sites with two-step assembly. (A) Two-step assembly works by assembling inserts first and then ligating the assembly reaction with the destination vector. Squares indicate restriction sites for BsaI (blue), BsmBI (red), AarI (yellow) and BbsI (green). (B) Domestication and characterization of promoters using level-0 promoter acceptor. (C) Domestication and characterization of terminators using level-0 terminator acceptor. T and CT terminators differ in the 5' end of the part (Supplementary Figure S2), with CT terminators including a stop codon. In B and C, fluorescence was normalized to OD (600 nm) and is shown as % of that of the J23100 promoter. Error bars indicate standard deviation, n = 9 (biological and technical triplicate).

Figure 5.

Use of secondary sites to screen variants of a transcription factor gene. (A) The parts used for the construction of the cI^ts library were: a constitutive promoter, an RBS+N-terminus part, cI^ts CDS without N or C terminus, and C-terminus + Terminator. The RBS+N-terminus part was PCR-built from a degenerate oligonucleotide variable for the Shine–Dalgarno consensus and for the codon following the start ATG, thus giving the library diversity in translation initiation and half-life of the protein (40). (B) Colonies that were white at 30°C were tested for expression of the mCherry reporter at 37°C. As a control, we analyzed the fluorescence of two colonies from the library that were unrepressed at 30°C (clones G4 and H1), two colonies with a constitutive mCherry gene, and four colonies from an equivalent library assembled with the wild-type (thermostable) cI CDS part.