Journal: Synthetic Biology
Article Title: Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology
Figure Lengend Snippet: 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 (G 1 to G n ) 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).
Article Snippet: Meanwhile, the destination vector is digested with AarI (Thermo Fisher Scientific Inc.) or BbsI-HF (NEB) and Shrimp Alkaline Phosphatase (NEB).
Techniques: Plasmid Preparation, Selection, Marker, Expressing, Conjugation Assay, Sequencing, Construct, Clone Assay