r0580  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

doi: 10.1093/nar/gkv466

Figure Lengend Snippet: Yeast Golden Gate (yGG) to assemble transcription units (TUs) flanked by VEGAS adapters. ( A ) yGG reactions to build TUs destined for VEGAS pathway assembly in S. cerevisiae include five parts: a left VEGAS adapter (LVA), a promoter (PRO), a coding sequence (CDS), a terminator (TER) and a right VEGAS adapter (RVA). Each part is flanked by inwardly facing recognition sequences for the BsaI restriction enzyme, an ‘offset cutter’ which cuts outside its recognition sequence (at positions 1/5 bp downstream) to expose the indicated four base-pair overhangs. All parts are cloned into vectors encoding kanamycin resistance (KAN R ) and an E. coli replication origin (Ori). ( B ) The yGG acceptor vector for VEGAS is designed such that outwardly facing BsaI sites expose overhangs corresponding to the 5′ LVA and 3′ RVA overhangs to promote assembly of the TU in the vector during a one-pot restriction-digestion reaction. The RFP cassette, built for expression in E. coli , is cut out of the vector when a TU correctly assembles, enabling white–red screening. The yGG acceptor vector encodes resistance to ampicillin (AMP R ) ( C ) The structure of a VA-flanked TU assembled by yGG. An assembled TU plus the flanking VA sequences may be released from the yGG acceptor vector by digestion with BsmBI.

Article Snippet: Terminal homology VEGAS ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

doi: 10.1093/nar/gkv466

Figure Lengend Snippet: VEGAS with adapter homology to assemble the carotenoid pathway in S. cerevisiae . ( A ) The four β-carotene pathway genes ( crtE, crtI, crtYB and tHMG1 ), assembled as TUs flanked by the indicated VAs (see Table 2 for PRO and TER parts), were released from the yGG acceptor vector with BsmBI digestion and co-transformed into yeast with the linearized VEGAS assembly vector. ( B ) S. cerevisiae colonies encoding assembled pathways develop a bright yellow color on medium lacking uracil (SC–Ura; left panel) as well as on YPD medium supplemented with G418 (right panel).

Article Snippet: Terminal homology VEGAS ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

Techniques: Plasmid Preparation, Transformation Assay

Journal: Nucleic Acids Research

Article Title: Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

doi: 10.1093/nar/gkv466

Figure Lengend Snippet: VEGAS with adapter homology to assemble a five-gene pathway. ( A ) The pathway consisting of VA-flanked TUs assembled by yGG may be released in one piece from the yGG acceptor vector by digestion with BsmBI (scissors). ( B ) A genetic pathway may be assembled into the linearized VEGAS assembly vector in S. cerevisiae by homologous recombination between VAs that flank TUs (TU1–5). X's indicate homologous recombination.

Article Snippet: Terminal homology VEGAS ∼1 μg of yGG-assembled, VA-flanked TU constructs were digested with BsmBI (New England Biolabs, R0580) in a final volume of 20 μl.

Techniques: Plasmid Preparation, Homologous Recombination