Noskov Vladimir N, Karas Bogumil J, Young Lei, Chuang Ray-Yuan, Gibson Daniel G, Lin Ying-Chi, Stam Jason, Yonemoto Isaac T, Suzuki Yo, Andrews-Pfannkoch Cynthia, Glass John I, Smith Hamilton O, Hutchison Clyde A, Venter J Craig, Weyman Philip D
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
ACS Synth Biol. 2012 Jul 20;1(7):267-73. doi: 10.1021/sb3000194. Epub 2012 May 21.
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 that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.
通过酵母重组组装大片段原核生物DNA的能力在合成生物学中具有重要应用，但在酵母中克隆大片段高G+C原核生物DNA可能具有挑战性。在酵母中克隆大片段高G+C DNA时的其他考虑因素可能与毒性基因、DNA大小或序列中缺乏酵母复制起点有关。作为我们在酵母中克隆高G+C DNA能力的一个例子，我们选择了聚球藻PCC 7942进行研究，其平均G+C含量为55%。我们确定该染色体的任何区域对酵母都没有毒性，并且超过约200 kb的聚球藻DNA片段不能稳定维持。总大小在200 kb以下的DNA构建体可以很容易地组装，即使片段之间有62 kb的重叠序列。在整个序列中添加酵母复制起点使我们能够将可组装的DNA总大小增加到至少454 kb。因此，利用酵母重组克隆大型高G+C原核生物序列的克隆策略应将酵母复制起点作为设计过程的一部分。
