Synthetic Genomics: Rewriting the Genome Chromosome by Chromosome Almer van der Sloot, Mike Tyers  Molecular Cell  Volume 66, Issue 4, Pages 441-443 (May 2017) DOI: 10.1016/j.molcel.2017.05.007 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Synthetic Genome Assembly Approaches (A) Schematic of Sc2.0 design rules depicting inserted, removed, and replaced parts. Top, wild-type chromosome; bottom, synthetic counterpart. (B) Genome synthesis and SwAP-In approach. Initial building blocks are synthetic DNA fragments (I). Building blocks are assembled into <10 kb chunks (I) and then into 30–60 kb megachunks containing a selectable auxotrophic marker (II). In the SwAP-In scheme (III), the first megachunk is integrated into the natural chromosome by HR. After auxotrophic selection of the integrated synthetic segment by marker A (green), the second megachunk overwrites marker A upon integration and selection by marker B (red). This procedure is iteratively repeated until all wild-type sequence is replaced by synthetic sequence. Following every megachunk incorporation, clones are checked by PCRtag analysis and assessed for fitness. (C) Contrasting strategy for Mycoplasma mycoides synthetic genome construction and transplantation to create JCVI-Syn1.0 strain (Gibson et al., 2010). Synthetic DNA fragments are assembled into progressively larger parts (I) and assembled in yeast into the full synthetic M. mycoides genome (II), which is then isolated and transformed in a Mycoplasma capricolum recipient species followed by elimination of the host genome (dashed circle) (III). (D) Parallelized SwAP-in-MRA scheme. SwAP-in of six to seven megachunks is carried out in multiple strains, then intermediate strains are crossed, and progeny harboring the full set of mega-chunks identified by PCRtags. Procedure is repeated until the entire wild-type chromosome is replaced by synthetic sequence. Two or more synthetic chromosomes are consolidated in a single poly-syn strain by an endoreduplication backcross. Molecular Cell 2017 66, 441-443DOI: (10.1016/j.molcel.2017.05.007) Copyright © 2017 Elsevier Inc. Terms and Conditions