Figure 2

Construction of a semi-synthetic M. mycoides genome by RMCE. (A) Schematic diagram of three steps of RMCE. First step: replacement of a 100 kb segment with the landing pad (red bar) via homologous recombination (broken arrows) in yeast. Second step: transformation of the donor plasmid pRC60 carrying the 100 kb synthetic segment (gray solid arrow) to the yeast containing the landing pad genome. Third step: introduction of the synthetic DNA segment into the genome via the loxP sites catalyzed by Cre recombinase, represented by purple arrows. (B) BssHII restriction enzyme maps of M. mycoides genomes:(I) Wild type, (II) the landing pad replacement, and (III) the semi-synthetic genomes. Three BssHII sites exist in wild type M. mycoides genome and two sites are overlapped with each other. The target 100 kb segment (blue arrow) in the genome (I) was replaced with the landing pad (red arrow) shown in genome (II) which was subsequently exchanged with the synthetic counterpart (green arrow) shown in genome (III). An additional BssHII site exists in the synthetic DNA segment, closest to the 3′end of the segment. (C) Contour-clamped homogeneous electric field (CHEF) electrophoresis analysis of BssHII-digested M. mycoides genomes. M. mycoides genome purified from yeast was digested with BssHII shown in the left panel, lane 1: wild type, lane 2: the landing pad replacement, and lane 3: the semi-synthetic genomes. M. mycoides genomes purified from bacterial transplants were digested with BssHII shown in the right panel, lane 1: wild type, lane 2: semi-synthetic clone 1 and lane 3: semi-synthetic clone 2. M: 50 kb lambda DNA ladder.