Fig. 1

Nanochromosome construction and engineering (also see Additional file 1: Fig. S1). (a) In vitro and in E. coli, we assembled a centromeric, K. phaffii ARS-containing, framework. We added an initial gene-integration array (landing zone) and proto-telomeres, creating nanochromosome (nChr)-precursor version (v)1. We cleaved this between its proto-telomeres, and used the linear product - nChr 1 - to transform K. phaffii cells. To improve nChr stability, we extended nChr-precursor v1 and added a second ARS (for precursor v2), then replaced the initial integration array with new ones, creating precursors v2A and v2B. These yielded nChr 2A and nChr 2B, which were stable only in a ΔKU70 strain. Finally we explored the feasibility of engineering nChr 2A and nChr 2B (to Chr 2A.1 etc.) in vivo. Plasmids are not drawn to scale. (b) Schematic representations (drawn approximately to scale) of the synthetic, linear nanochromosomes as constructed in the current study. Lists of oligos, plasmids and strains may be found in Additional file 2: Tables 1, 2 and 3 respectively. Promoters and terminators are not shown. CEN = centromere, ncDNA = non-coding DNA, LHR = long HR-compatible region, mCH = mCherry gene, GFP:FH = gene coding for a GFP-FH fusion