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. 2019 Apr 1;116(16):8070–8079. doi: 10.1073/pnas.1818259116

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Copyright © 2019 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 2. — Assembly of C. eth-2.0 in S. cerevisiae. (A) Schematic representation of the circular 785,701-bp C. eth-2.0 chromosome with six auxotrophic selection markers (red), 11 ARSs (black), and the restriction sites for PmeI and PacI (blue); 236 DNA blocks (green boxes) were assembled into 37 genome segments (blue boxes) and 16 megasegments (orange boxes) and further assembled into the complete C. eth-2.0 genome (outermost gray track). (B) The complete C. eth-2.0 chromosome was assembled in a single reaction from 16 megasegments by yeast spheroplast transformation and subsequent growth selection for auxotrophic TRP1 and LEU2 markers. (C) Growth selection on medium lacking Ura, Trp, His, Met, Leu, and Ade identified yeast clone 2 (C. eth-2.0) positive for all auxotrophic markers, while the parental strain (YJV04) fails to grow and requires synthetic defined (SD) medium. (D) Size validation of the 785-kb C. eth-2.0 chromosome by pulsed field gel electrophoresis. Digestion with PmeI and PacI releases a 771-kb portion of the C. eth-2.0 chromosome (arrow) from the shuttle vector pMR10Y. Undigested (marker) and PmeI- and PacI-digested yeast chromosomes (YJV04 digest) serve as controls. (E) DNA sequencing coverage at segment level (Top) and megasegment level (Middle) and the complete chromosome assembly (Bottom) are shown.