Mobius Assembly: A Versatile Framework For Golden Gate Assembly

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/140095_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (16K):\norg.highwire.dtl.DTLVardef@165d4e4org.highwire.dtl.DTLVardef@7bd8f1org.highwire.dtl.DTLVardef@dfbda4org.highwire.dtl.DTLVardef@11b8db9_HPS_FORMAT_FIGEXP M_FIG C_FIG Golden Gate Assembly is a powerful synthetic biology tool, which utilizes Type IIS enzymes for unidirectional assembly of multiple DNA fragments. The simplicity of its DNA assembly and the exchangeability of standard parts greatly facilitate the generation of combinatorial assembly libraries. Currently there are two popular Golden Gate Assembly frameworks that allow multigene augmentation (MoClo and Golden Braid); they render either high cloning capacity or vector toolkit simplicity. We have developed a new Golden Gate Assembly framework called Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. Mobius Assembly is based on a two-level approach and embraces the standard overhangs defined by MoClo and Golden Braid to confer exchangeability, but with reduced domestication requirement. Furthermore, we have implemented drop-out cassettes encoding chromogenic proteins for visible cloning screening. As proofs of concept, we have functionally assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements.