SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes

Abstract

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard—but not its entirety—has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

System metabolic engineering of Escherichia coli W for the production of 2-ketoisovalerate using unconventional feedstock

“…We assembled a synthetic expression system using a modular cloning technique based on Golden Standard ( Blázquez et al, 2022 ; Martínez-García et al, 2022 ). Simultaneous design of overexpression systems supported combinatorial, multi-part assembly of standardized genetic elements to deliver genetic circuits.…”

Technical upgrade of an open-source liquid handler to support bacterial colony screening

“…Next, we tested the screening capacity of COPICK based on color measurement. To do that, we prepared LB-agar plates supplemented with X-gal at 50 µg/ml, and inoculated them at proper dilution factor with a 50-50% mixture of E. coli DH5α carrying two pSEVA plasmids: pSEVA637 (colonies exhibit a white color) and pSEVA 6819[gD] (colonies exhibit a blue color in presence of X-gal) (Martínez-García et al, 2023). Based on a predefined target RGB color (corresponding to an arbitrary selection of blueish color displayed by pSEVA 6819[gD] strain), we programmed the selection of 64 clones exhibiting the most dissimilar color respect to the given reference in descending order (column-wise order), transferring them into a single-well plate (Nunc Omnitray, ThermoFisher Scientific).…”

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

“…In this case, DNA fragments were synthesized de novo to encode SpRY (nicking variant, carrying an additional D10A mutation), APOBEC1, 2UGI and ABEmax. Each of these DNA segments was codon-optimized to facilitate expression in Gram-negative bacteria and cured from restriction sites as outlined in the Standard European Vector Architecture (SEVA) framework [37,38]. The SEVA platform is a user-friendly plasmid system that facilitates standardization in synthetic biology, based on swapping and combining individual genetic modules (e.g.…”

“…It is and pS44i8GM (medium-copy-number) in P. putida cells after 10 h of growth (mid-exponential phase) with different concentrations of the inducer (0.5, 1 and 2 mM 3-mBz). Plasmid copy number was calculated by normalizing expression rates measured using qPCR of the plasmid-borne aadA gene (Str R ) and the chromosomal rpoB gene [29], and compared with the reference plasmid (pSEVA448 [38,43]).…”