Messerschmidt Sonja J, Kemter Franziska S, Schindler Daniel, Waldminghaus Torsten
LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-Universität Marburg, Germany.
Biotechnol J. 2015 Feb;10(2):302-14. doi: 10.1002/biot.201400031. Epub 2014 Nov 21.
Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications.
DNA组装方法的最新进展使合成染色体的合成成为一个可实现的目标。然而，对原染色体进行重新设计风险很高，且仍需要大量资源。另一种方法是向细胞中添加合成染色体。霍乱弧菌的天然次生染色体有可能作为大肠杆菌中合成次生染色体的模板。为了验证这一假设，我们基于霍乱弧菌染色体II（oriII）的复制模块构建了一个名为synVicII的复制子。基于对不稳定绿色荧光蛋白变体的流式细胞术分析，开发了一种评估复制子稳定性的新方法。将该方法应用于携带synVicII的细胞，结果显示与基于大肠杆菌oriC的次生复制子相比，其稳定性有所提高。细胞周期分析和synVicII细胞拷贝数的测定表明，大肠杆菌中合成复制子的复制时间与霍乱弧菌中的天然染色体II（ChrII）相当。本文展示的合成生物学工作为利用大肠杆菌中的次生染色体回答基础研究问题以及开展多种生物技术应用提供了基础。
