Figure 1

Workflow and sequencing method for library analysis. (A) Transformed libraries were handled one of two ways: (I) Sample proportions of MVT were assessed by plating serial dilutions on selective media yielding resolved colonies upon outgrowth (I.a). Genes from ~20 colonies were sequenced using an optimized, cell-templated protocol (I.b and I.c), and MVT were identified by overlapping peaks in their sequencing chromatograms. To estimate the average number of unique plasmids born by these MVT, ten verified MVT were restreaked to single cell resolution on fresh selective plates (I.d). Genes from ~10 colonies of each restreaked plate were sequenced (I.e and I.f), and the number of plasmids in the corresponding parental clone was estimated from the number of unique genes per plate. (II) In parallel experiments, the library proportion of MVT was assessed as function of outgrowth time. Following transformation, cells were grown with aeration in selective liquid media at 30°C (II.a). Every two hours, aliquots were plated yielding resolved colonies upon outgrowth (II.b). Eleven or more colonies from selected time points were assessed for the presence of multiple plasmids by DNA sequencing (II.c and II.d). (B) Top - chromatogram from a MVT colony exhibiting overlapping peaks (target bases 121-124). Note that the ABI base calling algorithm incorrectly identifies the third multiple peak as an "A". This underscores the importance of visual chromatogram inspections. Bottom - corresponding chromatogram from a monoclonal colony template.