SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities

Abstract

The Standard European Vector Architecture 2.0 database (SEVA-DB 2.0, http://seva.cnb.csic.es) is an improved and expanded version of the platform released in 2013 (doi: 10.1093/nar/gks1119) aimed at assisting the choice of optimal genetic tools for de-constructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA standard facilitates combinations of functional DNA segments that ease both the analysis and the engineering of diverse Gram-negative bacteria for fundamental or biotechnological purposes. The large number of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biology Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biology communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline.

Figure 1.

The formatted structure of SEVA plasmids. SEVA vectors are shaped by three basic modules: a cargo (blue), a plasmid replication origin (green) and an antibiotic marker (magenta). Restriction sites that punctuate the boundaries between modules in all constructs are indicated. Note the numbering position +1 of the DNA sequence is the first T of the unique PacI site. The directionality of the stronger transcription flow in the genetic device engineered in the cargo and the preferred site for inserting functional gadgets are indicated. See http://seva.cnb.csic.es for details.

Figure 2.

Expanded SEVA nomenclature. Vectors include four modules (antibiotic resistance marker, replication origin, cargo and gadget), which are represented in a code with four unequivocal positions. The first position is for antibiotic resistance markers, numbered 1 to 9 for the first nine and then 9B to 9Z for the next ones. The second position (the plasmid origin of replication) also receive a 1 to 9 code for the first variants and 9B to 9Z for those that follow. The cargo (third position), each cargo is assigned a sole number 1 to n, but the figure can be added with a capital letter in cases of variants of the same module. The fourth position is kept for the gadgets, which receive Greek letters (α to ω). See text for rationale and detailed explanation.

Figure 3.

Details of the translation of pSEVA111 into SBOL format. The image to the left represents the breakdown of pSEVA111 (inner circle) in different sections until every single base in the sequence is assigned a role. This process is done hierarchically from up (components) to bottom (subcomponents). We first separate the sequence into ‘SBOL annotations’ (enzyme targets in black and functional modules in light brown) and second divide them into subunits when possible (dark brown). Names of all parts are shown at the right side of the figure. Terminator ‘T1’ is considered the whole sequence that goes from AscI to PacI and it is composed of three subcomponents: scars ‘ScarT1a’ and ‘ScarT1b’ (bases that do not correspond to the terminator itself but are there due to assembly imperfections), and ‘T1seq.’ which is the T1 sequence (in SBOL the labels ‘T1’ and ‘T1seq.’ refer to well differentiated segments and cannot be named with the same tag). In the same way, the ‘Cargo’ section is divided into: primer sites ‘R24’ and ‘F24’ and the multiple cloning site ‘MCS’. Terminator T0 is formed by the original sequence ‘T0seq.’, a target for ‘SanDI’ and another scar, ‘ScarTO’. The SBOL description is finally serialized as an RDF/xml file (see Supplementary File S1), a sample of which is pasted at the bottom of the figure.

Figure 4.

The SEVA database with the SBOL extension. The goal of the sketched flowchart is the attachment of an SBOL file to each SEVA vector in the collection and the use of that information for managing a queryable database. Developers and administrators will contribute by inputting and storing data using three levels: the repository of SEVA plasmids, the repository of their corresponding SBOL descriptions and a composite list of both items. End users will be able to retrieve that information to use it in the wet laboratory as well as in a previous in silico design stage.