《Assembly of Large High G+C Bacterial DNA Fragments in Yeast》.pdf

Research Article /synthbio Assembly of Large, High G+C Bacterial DNA Fragments in Yeast Vladimir N. Noskov,† Bogumil J. Karas,‡ Lei Young,† Ray-Yuan Chuang,† Daniel G. Gibson,‡ Ying-Chi Lin,‡ Jason Stam, ‡ Isaac T. Yonemoto,‡ Yo Suzuki,‡ Cynthia Andrews-Pfannkoch,† John I. Glass, † Hamilton O. Smith,‡ Clyde A. Hutchison, III,‡ J. Craig Venter, ‡ and Philip D. Weyman*,‡ †Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, United States ‡Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 10355 Science Center Drive, San Diego, California 92121, United States S *Supporting Information ABSTRACT: The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ∼200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA