The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

Abstract

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Figure 1.

Representative search results of the SEVA-DB. The welcome page includes a header with active links to the formatted plasmid structure, plasmid backbone, modules, nomenclature, plasmid list and contact information. Typically, the user will browse through the plasmid list, where the collection of constructs are ordered according to their origin of replication, antibiotic selection marker, type of cargo and a SEVA code. The page for each plasmid also has active links to the corresponding GenBank number and the complete DNA sequence of each construct.

Figure 2.

Overall organization of structure of SEVA plasmids. (a) SEVA vectors are formed by three variable modules: a cargo (blue), a replication origin (green) and an antibiotic marker (magenta). Enzymes used to change the functional DNA segments are shown in the same color code and modules are separated by three permanent regions, which are shared by all vectors, the T₀ and T₁ transcriptional terminators and the oriT conjugation origin. All universal primers (PS1–PS6) are placed within the invariable backbone and are used to sequence/check the variable modules. (b) The structure of the default SEVA cargo. Cargos are cloned as PacI/SpeI fragments. The default segment contains the typical pUC18 polylinker enzymes from EcoRI to HindIII (the completely ordered restriction enzyme list is highlighted in the figure). Additional enzymes (i.e. SfiI, AvrII and NotI) are placed outside of the polylinker for specific cloning purposes. Within the cargo sequence, the enzyme recognition sites and the hybridization site of the universal M13 (R24/F24) primers are shown.

Figure 3.

SEVA nomenclature. Plasmid vectors are named using digits that reflect their functional modules. The codes include at least three positions: the first stands for antibiotic markers, the second for the replication origin and the third for the cargo. The lists of possible modules for each position are given, different versions of cargos are possible, and additional digits and letters are used to specify the modification (as shown in the lower right list).

Figure 4.

Organization of the edited SEVA plasmid replication origins. The R6K suicide origin lacks the gene encoding the Π replication protein, which makes its maintenance dependent on the trans delivery of that protein. RK2 is formed by the vegetative origin (oriV) and the replication protein trfA. pBBR1 has a similar structure to RK2; however, the pRO1600/ColE1 origin is a hybrid of the pRO1600 origin and the ColE1 replication sequence. For RSF1010, the mob genes have been eliminated and the synthetic sequence has only the oriV and the repBAC genes, which are expressed from a PlacUV5 promoter.

Figure 5.

Combination of expression and reporter cargos in the SEVA plasmids. (a) The structure of the default cloning cargo with the universal M13 primers (F24 and R24) and relevant restriction sites. (b) Assembling expression systems as PacI/AvrII fragments removes the R24 primer sequence and SfiI sites upstream of the AvrII site. (c) Cloning of reporter genes as HindIII/SpeI fragments removes the F24 primer sequence and a NotI site that is placed downstream of the HindIII site. (d) Combination of an expression system and reporter gene leaves an intact polylinker (from EcoRI to HindIII) and a unique NotI site between the AvrII and EcoRI sequences, which can be used to clone large constructs.