Figure 3

Stability of the duplication and phenotypes of the different derived genotypes. (a) Chromosomal map of the duplication in BW4005 showing primer pairs used to follow the fate of the duplication. The same symbols as in Figure 2 are used. (b) Example of PCR experiments to score the state of the genomic rearrangement, by using the three primer pairs IS5F1/IS5R1, IS3F1/IS3R1 and IS3F1/IS3R2. WT and DO stand for the presence of the single ancestral or 2668-bp deleted copy, respectively, and HD for the presence of the heterozygous duplication. The expected results for each of the three genotypes are shown in (c). * Owing to the higher efficiency of production of the smaller 0.5 kb PCR product with IS3F1/IS3R1, we had difficulties in detecting simultaneously the larger 3.1 kb PCR product in clones harboring the heterozygous duplication. We therefore also performed PCR reactions using the additional primer pair IS3F1/IS3R2 to improve the accuracy of scoring the heterozygous duplication state. (d,e) Stability of the heterozygous duplication under the selective conditions of the chemostat (d) and non-selective conditions of LB batch cultures (e). In (d), BW4005.C6 harboring the heterozygous duplication was mixed 1:99 with the MC4100 reference strain after individual acclimation in separate chemostats. Samples were taken every 24 hours for three days and appropriate dilutions were plated onto LB plates with or without 30 μg ml^-1 kanamycin. At each time point, we sampled 32 well-separated colonies from Kan-plates and scored them for the IS5-mediated duplication and IS3-mediated deletion. In (e), an overnight culture of BW4005.C6 was diluted 100-fold into LB liquid medium and grown for 24 hours. This growth cycle was repeated for two additional days and the proportion of the different genotypes was followed as in (b). The data presented are the average of two independent cultures in each case.