r3539  (New England Biolabs)

Journal: Synthetic Biology

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

doi: 10.1093/synbio/ysab003

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

Journal: Synthetic Biology

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

doi: 10.1093/synbio/ysab003

Figure Lengend Snippet: 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).

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, Fluorescence, Standard Deviation

Journal: Antibodies

Article Title: Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System

doi: 10.3390/antib10020018

Figure Lengend Snippet: Schematic illustration of BiDi promoter system for antibody production. ( A ) The MD vector was designed to exhibit a 200-bp stuffer, flanked by Esp 3I restriction sites ( Esp 3I sites A and B), adjacent to a SV40 polyA signal sequence and the regions encoding for hinge-CH2-CH3, terminated by a SV40 polyA signal sequence. ( B ) VL-CL and VH-CH1 amplicons can be inserted into MD by Golden Gate cloning utilizing Esp 3I restriction sites ( Esp 3I sites A and B). The BiDi promoter can be chosen individually and is flanked by Bbs I sites ( Bbs I sites A–D), compatible with the VL and VH sequences. ( C ) Golden Gate assembly results in a fully functional and re-circularized vector, with the light chain under the control of promoter I and the heavy chain under the control of promoter II. ( D ) Schematic representation of the resulting heterotetrameric IgG1 antibody using the same colour code as for the genetic elements.

Article Snippet: Assembly of the MD expression constructs was conducted with 75 ng destination vector and equimolar amounts of the respective fragments, 20 U Bbs I-HF, 10 U Esp 3I, and 200 U T4-DNA ligase (NEB, Frankfurt, Germany) for 30 cycles (1 min; 16 °C; 37 °C).

Techniques: Plasmid Preparation, Sequencing, Clone Assay, Functional Assay